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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Waxy solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot 40265/Lot 402678 20/80 ratio
- Expiration date of the lot/batch: 01 May 2017
- Purity test date: 09 Oct, 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent: Stable

FORM AS APPLIED IN THE TEST: Homogenized in arachis oil

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Male mean: 170 g, Female mean: 135.75 g
- Fasting period before study: None
- Housing: Grouped housed with 5 animals per sex in Macrolon cages
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 C
- Humidity (%): 40-70
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test article was dosed via oral gavage at at a volume of 5 mL/kg
Vehicle:
other: Arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on solubility information from the sponsor and data from previous studies that used oral dosing.
- Amount of vehicle (if gavage): All doses were administered at a volume of 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 6, and 13). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions and over 8 days in the refrigerator under protection from light were also determined (highest and lowest concentration, in Week 1).
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
15/sex, 5 males and 5 females reserved for the recovery group.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
10/sex
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
10/sex
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
15/sex. 5/sex reserved for the recovery groups. 1000 mg/kg/day group was not dosed from Day 31 onwards based on health status of the animals. The animals were weighed on Day 33 to evaluate if dosing could be re-started. Treatment of Group 4 was recommenced on Day 35 (and onwards) at a dose level of 600 mg/kg.
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
15/sex. 5/sex reserved for the recovery groups. 1000 mg/kg/day group was not dosed from Day 31 onwards based on health status of the animals. The animals were weighed on Day 33 to evaluate if dosing could be re-started. Treatment of Group 4 was recommenced on Day 35 (and onwards) at a dose level of 600 mg/kg.
No. of animals per sex per dose:
100, 300 mg/kg: 10/sex/dose. 0 and 1000-600 mg/kg: 15/sex/dose with 5 males and 5 females from each group reserved for recovery groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: Random
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
NA

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from the start of treatment onwards, detailed clinical observations were made in all animals after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4).

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes, weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At pretest and Week 13
- Dose groups that were examined: Control and high-dose groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of treatment (main and recovery animals) and at the end of recovery (recovery animals).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, max 24 hours
- How many animals: All
- Parameters checked: white blood cells, differential leucycyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distribution width, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of treatment (main and recovery animals) and at the end of recovery (recovery animals).
- Animals fasted: Yes, max 24 hours
- How many animals: All
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 12-13 of treatment
- Dose groups that were examined: Recovery group animals in the control and high-dose groups.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Tissues examined: adrenal glands, aorta, brain, caecum, cervix, clitoral gland, colon, duodenum, epididymides, eyes with optic nerve, Harderian gland, female mammary gland area, femur, heart, ileum, jejunum, kidneys, larynx, lacrimal gland, larynx, live,r lung, lymph nodes, nasopharynx, esophagus, ovaries, pancreas, Peyer's patches, pituitary gland, prostate gland, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina, all gross lesions.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals showed rales and swelling of the abdomen among all groups, showing higher incidence compared to controls at 300 and 1000/600 mg/kg in both sexes. Additional clinical signs at 1000/600 mg/kg included: hunched posture, labored or deep respiration and lean appearance. No clinical signs were noted during the recovery period and no additional findings were noted during the arena observations in this study. Salivation seen after dosing among all animals was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurence (i.e. after dosing). this sign was considered to be a physicological response related to taste of the vehicle and formulations rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were twelve premature decendents int eh study: 1/10 at 100 mg/kg, 1/10 at 300 mg/kg and 10/30 at 1000 mg/kg. Rales, swelling of abdomen and lean appearance were noted among these animals on the days prior to death.
Males at 1000/600 mg/kg (no. 36, 40, 47, 48, 50) and females at 1000/600 mg/kg (no. 88 and 100) were euthanized for ethical reasons after respectively 70, 19, 30, 62, 84, 22 and 63 days of treatment. Most of these animals showed body weight loss prior to death. Main cause of morbidity was hepatocellurlar necrosis of the centrilobula area of the liver and/or degenerative findings in the kidney.
Single female at 100, 300, and 1000 mg/kg (no. 74, 77, and 93) were found dead after respectively 91, 77 and 19 days of treatment. Although animals 74 and 77 showed body weight loss, no cause of death could be determined for all three animals for the sections examined.
One male and one female at 1000 mg/kg (no. 44 and 96) were found dead after respectively 26 and 30 days of treatment. Cause of death for these animals was a gavage accident. No body weight loss was noted in these animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male rats dosed with 1000/600 mg/kg test article began demonstrating a statistically significant reduction in mean body weight when compared to the control group on Day 8 which persisted throughout the dosing period with the mean body weight being 15% less than the control group by Day 91 of the study. Males dosed with 300 mg/kg demonstrated statistically significant reductions in mean body weights on Day 57 through 71 (-8 to -9%) and on Day 91 (=8%) while males dosed with 100 mg/kg demonstrated no differences in body weight over the course of the study. Female rats dosed with 1000/600 mg/kg demonstrated statistically significant reductions in mean body weight on test days 33 and 34 (-6 to -7%) when compared to the control group; however, these slight reductions were not considered adverse due to the sporadic and minimal nature of the change. No treatment-related body weight changes were noted in females in 100 mg/kg and females at 300 mg/kg. Following the 4-week recovery period, males dosed with 1000/600 mg/kg no longer demonstrated significant differences in body weight compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Minimal effects were observed in the food consumption data in males, with animals in the 1000/600 mg/kg group demonstrating a statistical reduction on Days 1-8 and elevated on Days 8-15, 29-57, 64-78 and 85-91. No alterations were in food consumption parameters were noted in males dosed with 100 or 300 mg/kg test article. Female rats dosed with 1000/600 mg/kg demonstrated statistically reduced food consumption on Day 1-8 and a statistical increase on Days 22-36, 43-57 and 78-85, which correlated with a significant reduction in relative food consumption on Days 1-8 and increased relative food consumption on Days 8-15, 22-57 and 67-91. Sporadic statistical increases in absolute and relative food consumption were noted in females from the 300 mg/kg dose group which were considered minimal in nature and non-adverse; females dosed with 100 mg/kg demonstrated no changes in food consumption parameters.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalomology findings were noted that were considred to be related to treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in hematology parameters distinguished treated from control animals:
- Lower lymphocyte levels in females at 300 and 1000/600 mg/kg.
- Higher white blood cell counts in females at 1000/600 mg/kg.
All effects recovered after a 28-day recovery period. The slightly lower red blood cells, higher reticulocytes and red blood cell distribution width, lower mean corpuscular volume and mean corpuscular hemoglobin in control females is only seen in the first 10 females which were sampled twice within a week. Therefore, the findings were considered to be a result of recovery after blood sampling and not related to the test item. Any other statistically significant changes in hematology parameters were considered to be unrelated to treatment as they occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical chemistry parameters distinguished treated from control animals:
- Higher alanine aminotransferase (ALAT) in males and females at 300 and 1000/600 mg/kg.
- Higher aspartate aminotransferase (ASAT) in females at 1000/600 mg/kg.
-Higher alkaline phosphatase (ALP) in males and females at 300 and 1000/600 mg/kg.
- Higher total bilirubin in males and females at 300 and 1000/600 mg/kg
- Higher bile acids in males at 1000/600 mg/kg
- Higher urea and creatinine levels in males at 300 and 1000/600 mg/kg
- Lower total protein in males and females at 300 and 1000/600 mg/kg.
- Lower glucose in males and females at 300 and 1000/600 mg/kg.
All effects recovered after a 28 day recovery period.

Any other statistically significant changes in clinical chemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static reflex were normal in all examined animals. Grip strength and motor activity was similar between control and high-dose animals. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly higher absolute liver weights were noted in both sexes at 300 and 1000/600 mg/kg and relative to body weight were noted in all treated males and females at 300 mg/kg and 1000/600 mg/kg. There was complete recovery in males and females following a 28 day recovery period.
Statistically significantly higher kidney weights (absolute and relative to body weights) were noted in all treated males. In females, kidney weights relative to body weight were increased in all groups and absolute kidney weights were increased in the 300 mg/kg and 1000/600 mg/kg groups. There was partial recovery for this increase in males (significant relative to body weight) and complete recovery in females at 1000/600 mg/kg after the 28 day recovery period.An increase in thyroid gland weight in males and females (relative to body weights) was noted starting at 300 mg/kg and 1000/600 mg/kg (only statistically significant increas of absolute thyroid gland weight at 300 mg/kg). There was partial recovery for this increase (apparent increase, not statistically significant) at 1000/600 mg/kg after the 28 day recovery period. Statistically significantly higher adrenal gland weights (abolustel and relative to body weights) were noted at 1000/600 mg/kg. There was complete recovery after the 28 day recovery period. An apparent decrease in thymus weight was noted in males and females at 300 mg/kg and 1000/600 mg/kg (only statistically significant increaese of absolute thymus weight at 300 mg/kg). There was complete recovery for this decrease at 1000/600 mg/kg after the 28 day recovery period. The thymus weight of the recovery females at 1000/600 mg/kg was statistically higher compared to the control recovery females. A statistically significant decrase in spleen weight (absolute and relative to body weights) was noted in all treated groups. There was complete recovery for this decrease at 1000/600 mg/kg after the 28 day recovery period.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Liver: An enlarged liver at the end of the treatment period was recorded in 2/10 males at 100 mg/kg, in 10/10 males and 3/9 females at 300 mg/kg and in 5/5 males and 6/6 females at 1000/600 mg/kg. After a 28-day treatment-free recovery period enlargement of the liver was not recorded. Discoloration (red-brown or black-brown) at the end of the treatment period was recorded 4/10 males and 3/9 females at 300 mg/kg and in 5/5 males and 6/6 females at 1000/600 mg/kg. After a 28 day treatment-free recovery period red-brown discoloration for the liver was recorded in 2/4 males and 2/5 females at 1000/600 mg/kg (microscopic correlate: brown pigment deposition). An accentuated lobular pattern in the liver was recorded in 1/10 males and 3/9 females at 300 mg/kg. This finding was not recorded after a 28-day treatment-free recovery period.
Kidney: Discoloration (red-brown or greenish) was recorded 1/10 males at 100 mg/kg, 3/10 males and 1/9 females at 300 mg/kg and 5/5 males and 5/6 females at 1000/600 mg/kg. After a 28 day treatment-free recovery period discoloration of the kidneys was recorded in 2/5 females at 1000/600 mg/kg.
Thyroid gland: An enlarged thyroid gland, was recorded in 2/10 males and 2/9 females at 300 mg/kg, in 1/6 main females at 1000/600 mg/kg and in the recovery group in 1/5 males of the control and 1/4 males of the 1000/600 mg/kg treated group. After a 28-day treatment-free recovery period enlarged thyroid glands were recorded at comparable incidences in control and treated rats.
Stomach: Macroscopic findings were recorded in all dose groups including controls. These findings consisted of dark red/reddish/black-brown foci in the glandular mucosa of the stomach in 1/10 males of Main Group 1, 1/5 males of the Recovery Group 1, 1/10 males and 2/9 females at 100 mg/kg, 1/5 males at 1000/600 mg/kg, reddish/black foci in the forestomach in 1/9 females at 100 mg/kg, 1/9 females at 300 mg/kg and 3/6 females at 1000/600 mg/kg and an irregular surface of the forestomach in 1/10 males at 100 mg/kg and 2/5 males at 1000/600 mg/kg. There was complete recovery for the stomach findings after a 28 day treatment-free recovery period. The macroscopic findings recorded at the end of the treatment period were considered to be related to the gavage treatment procedure with Arachis Oil with or without test item. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence and severity of hypertrophy of the follicular cells of the thyroid gland was recorded in males starting at 100 mg/kg and females at 1000/600 mg/kg. There was complete recovery for this finding after a 28 day treatment-free recovery period. The incidences and severities recorded for females at 100 and 300 mg/kg and the males and females of the recovery groups were within background pathology for rats of this age and strain.
Microscopic findings above background incidences and severities were recorded for the stomach (forestomach) of all dose groups including controls. These microscopic findings consisted of lymphogranulocytic inflammation, hyperplasia of squamous cells, erosions/ulcerations and edema. There was complete recovery for these findings in males and almost complete recovery in females after the 28-day treatment-free recovery period.
A combination of findings was recorded for the liver of males and females:
Centrilobular/diffuse hepatocellular hypertrophy with eosinophilic cytoplasm was recorded starting at 100 mg/kg. There was complete recovery for this finding after a 28-day treatment free recovery period. Hepatocellular necrosis of the centrilobular area (in some instances with additional brown pigmentation) was recorded in females starting at 300 mg/kg and in males at 1000/600 mg/kg. There was complete recovery this finding in males and partial recovery in females. Focal/multifocal coagulative necrosis was recorded at an increased incidence and severity in males at 1000/600 mg/kg. There was complete recovery for this finding after a 28 day treatment-free recovery period. The single incidences of minimal coagulative necrosis recorded in the remaining dose groups including the control recovery females is considered to be within background pathology of rats of this age and strain. Yellow-brown pigment deposition was recorded in a single male and a few females at 1000/600 mg/kg of the recovery group. A combination of findings was recorded for the kidney of males and females: Tubular basophilia was recorded at an increased severity in both sexes at 1000/600 mg/kg. There was complete recovery for this finding after a 28-day treatment-free recovery period. The incidences and severities recorded for the males and females of the remaining dose groups including controls were considered to be within background pathology for rats of this age and strain. Vacuolar degeneration/necrosis was recorded in one male surviving the 90-day treatment period. There was complete recovery for this finding after a 28-day treatment-free recovery period.
Granular casts were recorded in one male at 1000/600 mg/kg. There was complete recovery for this finding after a 28-day treatment-free recovery period. Eosinophilic content of the tubuli was recorded at an increased incidence and severity in males and females at 1000/600 mg/kg. There was complete recovery for this finding after a 28-day treatment-free recovery period. Eosinophilic content of the papil was recorded in a few females starting at 300 mg/kg. There was complete recovery for this finding after a 28-day treatment-free recovery period. Hyperplasia of the epithelium of the papil with cellular debris/casts was recorded in a few males and females at 1000/600 mg/kg. There was complete recovery for this finding after a 28 day treatment-free recovery period. A calculus in the papil or pelvis was recorded in a few females at 1000/600 mg/kg. There was complete recovery for this finding after a 28 day treatment-free recovery period. After a 28 day treatment-free recovery period yellow-brown tubular pigment was recorded in a few males at 1000/600 mg/kg.
Hypertrophy/hyperplasia of the urothelium of the urinary bladder was recorded in both sexes starting at 300 mg/kg. There was no recovery for this finding after a 28 day treatment-free recovery period.
An increased number of adipocytes (incidence and/or severity) in the bone marrow (sternum) was recorded in a few males and females starting at 100 mg/kg. There was partial recovery for this finding after a 28-day treatment-free recovery period.
A minor increase in incidence and severity of lymphocytolysis in the thymus was recorded in females starting at 100 mg/kg. There was complete recovery for this finding after a 28-day treatment-free recovery period.
A minor increase in incidence and severity of lymphocytolysis in the thymus was recorded in females starting at 100 mg/kg. There was complete recovery for this finding after a 28-day treatment-free recovery period.
A minor increase in incidence and severity of vacuolation of the zona glomerulosa of the adrenal gland was recorded in females at 1000/600 mg/kg. There was complete recovery for this finding after a 28-day treatment-free recovery period. The incidences and severities recorded for females at 100 and 300 mg/kg and the recovery groups were within background pathology for female rats of this age and strain.
A high incidence and severity of extramedullary hematopoiesis was recorded for the spleen of females of the Control group after the 90-day treatment period, compared to the test item treated dose groups. There was no extramedullary hematopoiesis in the spleen after the 28 day treatment-free recovery period in females of the control and 1000/600 mg/kg treated females, suggesting complete recovery. Remaining histologic changes were considered to be incidental. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) for the test article is 100 mg/kg/day.
Executive summary:

The subchronic toxicity potential of the test article was determined in male and female Wistar rats. The study was conducted in compliance with OECD GLP (1997). The test method was based on OECD 408. Male and female rats were dosed with 0 (15/sex with 5/sex designated for the recovery group), 100 (10/sex), 300 (10/sex) or 1000 (15/sex with 5/sex designated for the recovery group) mg/kg/day with the test article via oral gavage for 90 days followed by a 28 day recovery period. Based on clinical signs and general health of the animals treated at 1000 mg/kg, the dose level of this group was reduced to 600 m/kg from Day 35 onwards. Clinical signs (daily), body weights (weekly), food consumption (weekly), ophthalmoscopic examination (at Week 13), hematology and clinical chemistry (at the end of treatment for all animals and at recovery for recovery-group animals) were noted during the study. There were twelve unexpected decedents in the study. Five males and two females in the 1000/600 m/kg-treated groups were euthanized for ethical reasons during the treatment period. These animals demonstrated body weight loss and moribundity prior to death. The main cause of morbidity was hepatocellular necrosis of the centrilobular area of the liver and degenerative findings in the kidney. Single females at 100, 300, and 1000/600 m/kg were found dead during the treatment period. One male and one female at 1000/600 mg/kg were found dead during the treatment period. Cause of death for these animals was determined to be gavage accident. Notable clinical signs of toxicity noted in surviving males and females dosed with 1000/600 mg/kg test article were rales, swelling of the abdomen, hunched posture, labored or deep respiration and lean appearance at a higher incidence when compared to control group animals. At 300 mg/kg rales and swelling of abdomen were also noted at a higher incidence when compared to the control group. Male rats dosed with 1000/600 mg/kg test article began demonstrating a statistically significant reduction in mean body weight when compared with the control group on Day 8 which persisted throughout the dosing period with the mean body weight being 15% less than the control group by Day 91 of the study. Males dosed with 300 mg/kg demonstrated statistically significant reductions in mean body weights on Days 57 through 71 (-8 to 9%) and on Day 91 (-8%) while males dosed with 100 mg/kg demonstrated no differences in body weight over the course of the study. Female rats dosed with 1000/600 mg/kg demonstrated statistically significant reductions in mean body weight on test days 33 and 34 (-6 to -7%) when compared to the control group; however, these slight reductions were not considered adverse due to the sporadic and minimal nature of the change. No treatment related body weight changes were noted in females at 100 mg/kg and females at 300 mg/kg. Following the 4-week recovery period, males dosed with 600/1000 mg/kg no longer demonstrated significant differences in body weight when compared to the control group, indicating reversibility of this effect. Minimal effects were observed in the food consumption data in males, with animals in the 600/1000 mg/kg group demonstrating a statistical reduction on Days 1-8 and an elevation on Days 85-91. In conjunction with the body weight data, the relative food consumption in male rats dosed with 600/1000 mg/kg were statistically reduced on Days 1-8 and elevated on Days 8-15, 29-57, 64-78 and 85-91. No alterations were in food consumption parameters were noted in males dosed with 100 or 300 mg/kg test article. Female rats dosed with 600/1000 mg/kg demonstrated a statistically reduce food consumption on Days 1-8 and a statistical increased on Days 22-36, 43-57 and 78-85 which correlated with a significant reduction in relative food consumption on Days 1-8 and increased relative food consumption on Days 8-15, 22-57 and 67-91. Sporadic statistical increases in absolute and relative food consumption were noted in females from the 300 mg/kg dose group which were considered minimal in nature and non-adverse; females dosed with 100 mg/kg demonstrated no changes in food consumption parameters.

No effects were seen in the functional observations nor in the ophalmoscopy.

Postmortem examination of the animals revealed test article-related findings in the liver (hepatocellular necrosis of the centrilobular area and focal/multifocal coagulative necrosis and hepatocellular hypertrophy), kidney (vacuolar degeneration/necrosis and the granular casts in males and tubular basophilia in females), urinary bladder (hypertrophy/hyperplasia of the urothelium), thyroid gland (follicular cell hypertrophy), sternal bone marrow (increased number of adipocytes), stomach (lymphogranulocytic inflammation, hyperplasia of squamous cells, erosions/ulcerations and edema) in both male and female rats as well as changes in the thymus (lymphocytolysis), adrenal gland (vacuolation of the zona glomerulosa)and spleen (extramedullary hematopoiesis) of females.

In the liver the hepatocellular necrosis of the centrilobular area and focal/multifocal coagulative necrosis were degenerative findings and therefore considered adverse in nature. The minimal or slight hepatocellular hypertrophy of the liver observed in the males and females at 100 mg/kg, in the absence of any degenerative findings or changes in absolute liver weight was considered to be a nonadverse. Macroscopic correlates that were noted in these animals were discolouration, enlargement and accentuated lobular pattern of the liver. These microscopic findings correlated with increased serum liver enzymes, total bilirubine, bile acids and decreased total protein, glucose and cholesterol at 300 and 1000/600mg/kg test article.

In the kidney the vacuolar degeneration/necrosis and the granular casts recorded in males and the high severity of tubular basophilia observed in females were degenerative in nature and therefore considered to be adverse microscopic findings at 1000/600 mg/kg. These renal findings were accompanied by higher kidney weights and increased serum urea and creatinine levels in males at this dose level. The hypertrophy/hyperplasia of the urothelium of the urinary bladder in male and female rats dosed with 300 or 1000/600 mg/kg, where present at a low incidence, but demonstrated a dose-response relationship in severity and demonstrated no recovery and was, therefore, considered to be an adverse microscopic finding. Thyroid gland hypertrophy in rats is usually an adaptive response to induction of hepatic enzymes. This results in increase in the hepatic/biliary clearance of T3/T4 leading to increase in TSH and compensatory follicular cell hypertrophy and/or hyperplasia and is therefore, considered to be a secondary result of test article hepatic toxicity.

The findings reported in the sternal bone marrow, as well as thymus and adrenal observations in the female rats were considered to be spontaneous background findings as they demonstrated no dose-response relationship, were not accompanied by any degenerative findings and showed complete or partial recovery. Therefore these findings are considered to be non-adverse. Macroscopic and microscopic findings were recorded for the stomach of all dose groups including controls. There was no dose response relationship and therefore these findings were considered to be due to the gavage treatment procedure with Arachis Oil as vehicle and not related to the treatment with the test article.

The differences in severity of extramedullary hematopoiesis noticed in the spleen of females of all test item-treated dose groups compared to the control females at the end of the treatment period were considered to be related to the blood sampling procedure: Blood samples were collected from the females of the Main Control group after 92 days of treatment and these animals (except animal 51) were subsequently necropsied after 96 days of treatment on Day 97. No blood samples were collected of the remaining females on Day 92. The differences in the red blood cell parameters (decreased red blood cell counts and increased reticulocytes), increased organ weight of the spleen and microscopic finding (increased extramedullary hematopoiesis) in the spleen of the Main Control females after the treatment period, represent a physiological response after the blood sampling procedure and these differences between the control and test item treated groups are therefore regarded to be unrelated to the test item.

Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) of the test article is 100 mg/kg/day.