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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:
14 Jul 2014 to 01 Jul 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1998
Deviations:
yes
Remarks:
At the initiation of treatment animals were 9-10 weeks old, which is outside the recommended age of the OECD 408 guideline, which states before 9 weeks.
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.6200 (1998) Neurotoxicity screening battery
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD 417 (2010) Toxicokinetics
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
bis((1S,2R,6R,7S,8R)-11-(dichloromethylidene)-6-hydroxytricyclo[6.2.1.0²,⁷]undecan-3-one)
EC Number:
941-628-3
Cas Number:
1263184-87-7
Molecular formula:
C12H14Cl2O2
IUPAC Name:
bis((1S,2R,6R,7S,8R)-11-(dichloromethylidene)-6-hydroxytricyclo[6.2.1.0²,⁷]undecan-3-one)
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han Wistar designation Crl:WI (Han)
Details on species / strain selection:
The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 209-293 g (males) and 161-210 g (females)
- Fasting period before study: None
- Housing: 2 per cage by sex in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings which were provided with a certificate of analysis for significant contaminants.
- Diet: SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded was provided ad libitum (except during designated procedures)
- Water: From public supply ad libitum (except during designated procedures)
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 40-68
- Air changes (per hr): Minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
14 Jul 2014, end date not reported

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% (w/v) in Milli-Q water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The control substance, 1% (w/v) Carboxymethylcellulose (medium viscosity) in Milli-Q water, was prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily.

Test substance dosing formulations were prepared by adding the required amount of test substance to the vehicle and stirred magnetically. Once mixed, the formulation was then stirred using a high shear mixer until a homogeneous suspension was visually confirmed. The dosing formulations were prepared as often as required by stability, stored in a refrigerator set to maintain 4°C, and dispensed daily.

Control formulations were stirred for at least 30 min before and continuously during dose administration. Test substance formulations were stirred for at least 1 h before and continuously during dose administration.

VEHICLE
- Concentration in vehicle: 3, 10 and 100 mg/mL, corresponding to 30, 100 and 1000 mg/kg bw/day.
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analysis performed in a previous study demonstrated that the test substance is stable in the vehicle when prepared and stored under the same conditions bracketing those used in the present study. Duplicate samples (2 x 0.2 mL) were taken from the top middle and bottom of each formulation prepared for dosing during weeks 1, 6 and 12, then stored in a refrigerator at 4°C. Triplicate samples (3 x 0.2 mL) were also taken as backup samples and stored at 4°C. Samples were analysed by UPLC with uv detection within one week of preparation.
Duration of treatment / exposure:
92 days
Frequency of treatment:
Daily, seven days a week.
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Group 2, low-dose
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 3, mid-dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4, high-dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on results obtained in a previous study (Charles River Study No. 526295) where rats were dosed continuously for 28 days at dose levels of 0, 100, 300 or 1000 mg/kg/day. This resulted in histopathological findings of hepatocyte hypertrophy in the liver with associated increases in liver weight in males at 300 mg/kg/day and both sexes at 1000 mg/kg/day and thyroid gland follicular cell hypertrophy in males at 300 or 1000 mg/kg/day. In an attempt to produce graded responses to the test substance, the high dose level was expected to produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low dose level was expected to produce no observable indications of toxicity.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: (for general health/mortality and moribundity)
- Time schedule: Twice daily, am and pm

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Weekly, beginning Week -1.

POST DOSE OBSERVATIONS:
- Time schedule: Regularly throughout the day, particularly during the first four hours after dosing

BODY WEIGHT:
- Time schedule for examinations: Weekly from pretreatment until the completion of treatment. Body weight was also recorded on the first day on scheduled necropsy.

FOOD CONSUMPTION:
- Time schedule for examinations: Weekly from pretreatment until the completion of treatment.

WATER CONSUMPTION:
- Time schedule for examinations: Monitored by visual inspection of the water bottles on a regular basis throughout the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Once pre-dose and during week 13.
- Dose groups that were examined: All pre-dose, controls and high dose only week 13

HAEMATOLOGY:
- Time schedule for collection of blood: Prior to termination
- Sample taken from: Orbital sinus
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: All
- Parameters examined: Red blood cell count, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin concentration, Mean cell haemoglobin, Reticulocytes, Reticulocyte count (absolute), Red blood cell distribution width, Platelets, White blood cell count, Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Large unstained cells, Activated partial thromboplastin time, Fibrinogen, Prothrombin time.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Prior to termination
- Sample taken from: Orbital sinus
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: All
- Parameters examined: Urea, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Bile acids, Sodium, Potassium, Chloride, Triglycerides, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium, Inorganic phosphate.

URINALYSIS:
- Time schedule for collection of urine: during week 13 over a 4-6 hour period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Microscopic evaluation of spun deposit, Colour, Turbidity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Leukocytes, Blood pigments, Urobilinogen

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Once during week 12
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity:
Cage side observations :
• Posture/condition on first approach (animal undisturbed), checking for: Prostration; Lethargy; Writhing; Circling; Breathing abnormalities; Gait abnormalities; Tremor; Fasciculation; Convulsions; Biting (of cage components or self mutilating); Vocalisations; Piloerection.
• Ease of removal from the cage.
• Body temperature: a rectal temperature was taken.
• Condition of the eyes, checking for: Pupillary function; Miosis; Mydriasis; Exophthalmos; Encrustation; Lachrymation.
• Condition of the coat.
• Presence of salivation.
• Overall ease of handling.

Observations in a standardised arena (2 min):
• Latency (time to first locomotory movement).
• Level of mobility.
• Rearing.
• Grooming.
• Urination/defecation.
• Arousal (level of alertness).
• Posture, tremor/convulsions, vocalisation, piloerection.
• Palpebral closure.
• Gait abnormalities.
• Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

Functional tests: The following additional functional tests were performed for all animals during Week 12.
• Reaction to sudden sound (click above the head)
• Reaction to touch on the rump with a blunt probe
• Grip strength (fore and hind limbs)
• Pain perception (tail flick)
• Landing Foot Splay
• Motor activity
• Other physical/functional abnormalities: any other abnormality not already recorded in the above screening battery.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals)
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
- Organs weighed (all animals): adrenal glands, brain, epididymides, heart, kidneys, liver, seminal vesicles, ovaries, spleen, testes, thymus, uterus, prostate, thyroid.

HISTOPATHOLOGY: Yes (All tissues from all animals in the Control and High dose groups, gross lesions, liver and thyroid were examined histologically).
- Tissues examined: aorta, bone marrow (femur and sternum), bone (femur and sternum), brain, cervix, epididymides, eye, adrenal glands, mammary glands, Harderian glands, lachrymal glands, parathyroid glands, pituitary gland, prostate gland, salivary glands (mandibular), seminal vesicles, thyroid glands, Peyer's patches, heart, kidneys, large intestine (caecum, colon, rectum), larynx, liver, lungs, lymph nodes (mandibular, mesenteric), skeletal muscle, nasal cavity, optic nerves, sciatic nerves, oesophagus, ovaries, oviducts, pancreas, pharynx, skin, small intestine (duodenum, ileum, jejunum), spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, trachea, ureters, urinary bladder, uterus, vagina, abnormal tissue.
- An internal peer review was undertaken by an appropriately qualified veterinary pathologist, who validated the conclusions of the Study Pathologist, by independently assessing the microscope slides.
- An external peer review was conducted by the Sponsor's pathologist, any changes resulting from this have been retained in the study data.
Other examinations:
BONE MARROW SMEAR: Two prepared per animal but not examined.
LIVER SAMPLING: After samples were taken for histopathology, multiple samples of liver (12 x ca 150 mg) were taken from two 5 mm sections of the left lateral lobe, snap frozen in liquid nitrogen in individual RNA-ase free tubes and stored at -80°C pending possible future analysis. A representative section of the median lobe was taken, fixed in formalin and processed to paraffin wax block. A small piece of duodenum was incorporated into each block. The remainder of the liver was cut into chunks (approximately 2 g), snap frozen in liquid nitrogen and stored at -80°C.
Statistics:
All analyses were two-tailed for significance levels of 5% and 1%. If the variances were clearly heterogeneous, appropriate transformations (e.g. log, square root, double arcsine) were used in an attempt to stabilise the variances. Body weights, cumulative body weight gain, food consumption, selected functional observation battery & motor activity, absolute organ weights, haematology coagulation, clinical chemistry and selected urinalysis parameters were analysed initially by a one-way analysis of variance (ANOVA). Organ weights were also analysed by analysis of covariance (ANCOVA) on terminal body weight. This statistical analysis provided an Adjusted Organ Weight value. For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant. Micropathology incidence data was analysed using Fisher’s Exact Test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical observations that were considered to be treatment related. All clinical observations were considered to be typical of those seen on studies of this duration.
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:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no ophthalmic findings that were considered to be related to treatment with the test substance.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
White blood cell count, neutrophils and lymphocytes were lower in all male groups receiving the test substance when compared with controls. However, due to the nature of the change, no dose relationship and the lack of any correlating microscopic findings these were considered not to be treatment related.
Prothrombin time was slightly longer in males receiving 1000 mg/kg bw/day when compared with controls. However, as there were no changes noted in other related parameters and all but 2 of the individual values were within the control range this was considered incidental to treatment.
There were other differences from controls, some of which attained statistical significance, however, due to their isolated nature, the lack of a dose relationship and no correlating microscopic findings they were considered to be incidental to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Globulins were statistically significantly higher in males receiving 1000 mg/kg/day when compared with controls. This resulted in a statistically significantly higher total protein level and lower albumin/globulin ratio, when compared with controls. Albumin was statistically significantly higher in females receiving 1000 mg/kg/day when compared with controls. This also resulted in statistically significantly higher total protein, however, the albumin/globulin ratio was not affected when compared with controls.
Calcium levels were statistically significantly higher in animals receiving 1000 mg/kg/day when compared with controls. In addition, chloride levels were statistically significantly lower in females receiving 1000 mg/kg/day when compared with controls. Total bilirubin and cholesterol levels were higher in males receiving 1000 mg/kg/day when compared with controls.
Total bile acid levels were lower in all male groups receiving the test substance and females receiving 1000 mg/kg/day when compared with controls, with statistical significance being achieved in males receiving 100 or 1000 mg/kg/day. In the absence of associated histopathological findings the toxicological significance of this finding is uncertain.
Glucose levels were lower in all male groups receiving test substance when compared with controls. However, as the majority of individual values in the treated groups were within the concurrent control range this difference is therefore considered to be incidental and unrelated to treatment. There were no other treatment related effects.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary volume was higher in all male groups receiving test substance and females receiving 100 or 1000 mg/kg/day when compared with controls.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Tail flick was noted to be statistically significantly higher in females receiving 30 mg/kg/day when compared with controls. However due to the lack of a dose relationship and the variable nature of this parameter this was considered not to be related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and covariate liver weight was statistically significantly higher at 1000 mg/kg/day when compared with controls. In addition, absolute and covariate liver weight was slightly higher in females at 100 mg/kg/day when compared with controls, with statistical significance being achieved following covariate analysis. The increase in liver weight was associated with microscopic findings in the liver.
Absolute and covariate kidney weights were higher in males at 1000 mg/kg/day, with statistical significance being achieved following covariate analysis.
Absolute and covariate spleen weight was statistically significantly lower in males at 1000 mg/kg/day, however, due to the lack of any correlating haematological or microscopic findings these were considered not toxicologically significant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal to mild hepatocyte hypertrophy, either zonally (centrilobular) or diffuse (with loss of hepatocellular plate architecture), was observed in the liver in males and females at 100 mg/kg/day and above. When centrilobular and diffuse findings were combined, this reached statistical significance in both sexes at 1000 mg/kg/day (p ≤ 0.01 in males and p ≤ 0.05 in females). This also reached statistical significance in 100 mg/kg/day females (p ≤ 0.05). The liver hypertrophy in the high dose animals was associated with increased liver weights.
Minimal to mild follicular cell hypertrophy was observed in the thyroid gland in males at 100 mg/kg/day and above. When minimal and mild findings were combined for the follicular cell hypertrophy in the thyroid this reached statistical significance in males at 1000 mg/kg/day only (p ≤ 0.05).
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 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.:
1 000 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Analysis of formulations:

Analysis of formulations showed all groups to be within -3.3 to 2.0% of the theoretical concentrations and a coefficient of variation up to 4.1% indicating satisfactory accuracy of concentration and homogeneity.

Table 1: Intergroup comparison of selected clinical chemistry parameters

Parameter

Dose level (mg/kg/day)

 

Males

Females

0

30

100

1000

0

30

100

1000

globulin (g/L)

19

20

20

23**

17

16

16

17

albumin (g/L)

42

43

43

43

49

51

49

53*

total protein

62

63

63

66**

65

67

65

70*

A/G ratio

2.2

2.2

2.2

1.8**

3.0

3.1

3.0

3.1

calcium (mmol/L)

2.53

2.55

2.49

2.60*

2.49

2.51

2.51

2.58*

chloride (mmol/L)

102

102

103

101

104

104

104

103*

total bilirubin (mmol/L)

2.5

2.5

2.7

3.2**

2.5

2.5

2.5

2.5

cholesterol (mmol/L)

1.4

1.5

1.4

1.8**

1.2

1.4

1.2

1.4

glucose (mmol/L)

11.09

9.26*

9.44

9.15*

9.02

8.63

8.17

8.24

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 2: Intergroup comparison of urinary volume

Parameter

Dose level (mg/kg/day)

 

Males

Females

0

30

100

1000

0

30

100

1000

volume (mL)

0.8

1.8**

1.5

1.4

1.2

1.5

2.9*

2.8*

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 3: Intergroup comparison of selected organ weights (g)

 

Dose level (mg/kg/day)

Males

Females

organ

0

30

100

1000

0

30

100

1000

Liver

actual

12.79

13.13

12.99

15.12*

8.45

8.91

9.07

10.10**

 

covariate

12.51

12.88

13.44

15.20**

8.40

8.74

9.14*

10.25**

Kidney

actual

2.20

2.29

2.28

2.42

1.53

1.61

1.61

1.67

 

covariate

2.16

2.25

2.34

2.43**

1.52

1.58

1.62

1.69**

Spleen

actual

0.65

0.62

0.58

0.54*

0.46

0.45

0.47

0.44

 

covariate

0.64

0.61

0.59

0.54*

0.46

0.44

0.48

0.45

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 4: Summary of microscopic findings

 

Dose level (mg/kg/day)

Males

Females

organ

0

30

100

1000

0

30

100

1000

Liver / hepatocyte hypertrophy

no. examined

10

10

10

10

10

10

10

10

minimal

0

0

1

7

0

0

5

5

mild

0

0

0

0

0

0

0

1

total

0

0

1

7

0

0

5

6

Thyroid / follicular cell hypertrophy

no. examined

10

10

10

10

10

10

10

10

minimal

0

0

1

1

0

0

0

0

mild

0

0

0

4

0

0

0

0

total

0

0

1

5

0

0

0

0

 

Applicant's summary and conclusion

Conclusions:
Administration of the test substance once daily for at least 13 weeks by oral gavage was well tolerated in rats at levels of 0, 30, 100 or 1000 mg/kg/day, where hepatocyte hypertrophy in the liver correlating with higher liver weights and follicular cell hypertrophy in the thyroid gland were noted at 100 and/or 1000 mg/kg/day. In addition, changes in protein levels, calcium, total bilirubin and cholesterol were noted. Kidney weights were higher in males that received 1000 mg/kg/day and urine volumes were higher in all male groups and females that received 100 or 1000 mg/kg/day. Based on the above results and the fact that the highest severity level for the histopathological findings at 100 mg/kg/day was minimal, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg/day.
Executive summary:

In a GLP compliant subacute toxicity study according to OECD 408 the test substance was orally dosed, by gavage, to 4 groups of 10 male and 10 female Crl:WI(Han) rats for 13 weeks at dose levels of 0 (control), 30, 100 and 1000 mg/kg/day. The test substance was dosed using 1% (w/v) carboxymethylcellulose (medium viscosity) in Milli-Q water as the vehicle at a constant dose volume of 10 mL/kg. The animals were monitored regularly for viability and for signs of ill health or reaction to treatment. Body weights and food consumption were measured and recorded at predetermined intervals from pretreatment up to the completion of treatment. Functional observation battery assessments (including motor activity) were carried out on all animals in week 12. Blood samples were collected from all animals on the day of termination for haematology, coagulation and clinical chemistry parameters and urine samples were collected from all animals during week 13 for urinalysis parameters. After completion of treatment, all animals were euthanised and subjected to a detailed necropsy examination, where selected organs were weighed and tissues were preserved. Tissues from all animals in the Control and High dose groups and the liver, thyroid and gross lesions from low and intermediate groups were subjected to a histopathological examination.

Dose formulations were found to be accurately prepared and homogeneous at all sampling occasions. There were no treatment related clinical observations noted and no differences in body weight or body weight change, food consumption, ophthalmoscopy, haematology, clinical chemistry or functional observation battery measurements, including motor activity. Changes were noted in total protein, albumin globulin ratio, calcium, total bilirubin and cholesterol in males and/or females receiving 1000 mg/kg/day. Urinary volume was higher in all male treated groups and females receiving 100 and 1000 mg/kg/day. Higher kidney weights noted in animals that received 1000 mg/kg/day and females that received 100 mg/kg/day when compared with controls. Microscopic findings of hepatocyte hypertrophy in the livers of both sexes were noted at 100 and/or 1000 mg/kg/day. Follicular cell hypertrophy in the thyroids was observed in males only at 100 and/or 1000 mg/kg/day. Higher liver weights in males and females at 1000 mg/kg/day were considered to be associated with the microscopic findings.

In conclusion, administration of the test substance once daily for at least 13 weeks by oral gavage was well tolerated in rats at levels of 0, 30, 100 or 1000 mg/kg/day, where hepatocyte hypertrophy in the liver correlating with higher liver weights and follicular cell hypertrophy in the thyroid gland were noted at 100 and/or 1000 mg/kg/day. In addition, changes in protein levels, calcium, total bilirubin and cholesterol were noted. Kidney weights were higher in males that received 1000 mg/kg/day and urine volumes were higher in all male groups and females that received 100 or 1000 mg/kg/day. Based on the above results and the fact that the highest severity level for the histopathological findings at 100 mg/kg/day was minimal, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg/day.