N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

The 90-day repeated dose toxicity study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January 2015 to 05 February 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

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

EC Number:

941-634-6

Cas Number:

1228284-78-3

Molecular formula:

C10H12Cl3NO

IUPAC Name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

Test material form:

other: liquid

Details on test material:

- Physical state: Brown liquid
- Expiration date of the lot/batch: Recertification date end September 2015
- Storage condition of test material: Room temperature (<30°C), protected from light and humidity

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England
- Females nulliparous and non-pregnant: yes
- Age at study initiation: five to six weeks
- Weight at study initiation: On the first day of dosing the males weighed 173 g to 228 g and the females weighed 127 g to 171 g.
- Fasting period before study: No
- Housing: in groups of five, by sex, in grid-floor cages suspended over paper-lined trays.
- Diet (e.g. ad libitum): A pelleted diet, LabDiet 5L0S EURodent Diet (manufactured by PMI Nutrition International) supplied by International Product Supplies was freely available.
- Water (e.g. ad libitum): mains tap water (in bottles) was freely available.
- Acclimation period: seven days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C to 23 °C
- Humidity (%): 40 % to 70 %
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

by gavage using a rubber catheter and disposable syringe.

Vehicle:

other: aqueous 1 % (w/v) carboxymethylcellulose with 1 % (v/v) Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was formulated at intervals within known stability for dosing as a suspension in the vehicle, aqueous 1 % (w/v) carboxymethylcellulose with 1 % (v/v) Tween 80. The required amount of test item was weighed into a container and approximately 90 % of the final preparation volume of vehicle was added and then stirred. The formulation was heated to a recorded maximum of 48.9 °C until a homogeneous mixture was formed and was then allowed to cool to ambient temperature, before being transferred to a stoppered measuring cylinder and made up to final volume with vehicle. This was mixed by gentle inversion before being divided into daily aliquots in amber glass bottles, which were stored refrigerated (2 °C to 8 °C), before use. Test item formulations were stirred for at least 15 minutes before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations prepared at concentrations of 0.5 mg/mL to 25 mg/mL, spanning those used in this study (2 mg/mL to 15 mg/mL), have been shown to be stable for up to six days when stored at room temperature, up to 12 days when stored refrigerated and for one month when stored frozen.
Samples were taken from each formulation. Formulations prepared for use on Day 1 were analysed to assess their homogeneity and achieved concentrations. Having satisfactorily confirmed homogeneity on Day 1, subsequent formulations for use during Weeks 6 and 13 were analysed to determine achieved concentrations only. Samples from vehicle used to dose Controls on Day 1 and during Weeks 6 and 13 were analysed to confirm absence of test item. All samples were analysed for the substance using a validated method.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected in consultation with the Sponsor on the basis of results from a previous 28 day study in the rat

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed once weekly, starting pre-dose, for their behaviour both within their cage and then after placement in an open arena. Observations were made at approximately the same time of day on each occasion (afternoon).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination daily. In addition, for the first four weeks of treatment, animals were observed before, shortly after and about one hour after dosing. On weekdays a final check was made around four hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed daily for the first two weeks of treatment and then weekly until necropsy.

FOOD INTAKE: Yes
The amount of food consumed by each cage of animals was recorded daily for the first week of treatment and then weekly until necropsy.


OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes of all animals were examined before the start of treatment. Subsequently, all animals from each of the Control and high dose groups were examined in Week 12. Since unusual findings were seen in high dose females, intermediate dose females were also examined in Week 12, but as there were no unusual findings in these animals examination was not further extended to low dose females.
Examinations were performed using direct and indirect ophthalmoscopy after previous use of a mydriatic agent.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken from the sublingual vein of all animals during Week 13.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: all
- Parameters examined: haemoglobin concentration (Hb), platelet count (Plate), red blood cell count (RBC), total leucocyte count (WBC), packed cell volume (PCV), neutrophil (Neut), mean cell volume (MCV), lymphocytes (Lymph), mean cell haemoglobin (MCH), monocytes (Mono), mean cell haemoglobin concentration (MCHC), eosinophils (Eosin), reticulocytes (Retics), basophils (Baso), cell morphology, large unstained cells (LUC), red blood cell distribution width (RDW).


COAGULATION: Yes
- Time schedule for collection of blood:
Blood samples were taken from the sublingual vein of all animals during Week 13.
- Animals fasted: No
- How many animals: All
- Parameters examined.: prothrombin time (PT), activated partial thromboplastin time(APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the sublingual vein of all animals during Week 13.
- Animals fasted: No
- How many animals: All
- Parameters examined.: urea, albumin (Alb), creatinine (Cren), globulin (Glob), glucose (Gluc), albumin/globulin ratio (A/G ratio), alkaline phosphatase (ALP), cholesterol (Chol), alanine aminotransferase (ALT), triglycerides (Trigs), aspartate aminotransferase (AST), calcium (Ca), total protein (T. Prot), sodium (Na), total bilirubin (BiliT), potassium (K), gamma glutamyl transpeptidase (GGT), chloride(Cl), inorganic phosphorus (I. Phos), bile acids(B. Acids).

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
On one occasion during Week 12 of the treatment period, sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity were recorded for all animals.

IMMUNOLOGY: No


TOXICOKINETICS: Yes
- Time schedule for collection of blood: Blood samples (0.1 mL) were taken from the tail vein into K2EDTA anticoagulant. The lowest numbered four males and four females from each group were sampled on Day 90, pre-dose and at 30 minutes, 1, 2, 4, 8 and 24 hours after dosing.
Blood samples were taken from the sublingual vein of all animals during Week 13.
- Animals fasted: No
Only samples taken from test item-treated animals were analysed. Concentrations of the test item were determined using a validated LC-MS/MS method.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the treatment period all animals were killed by exposure to carbon dioxide gas in a rising concentration. Equal numbers of males and females from each group were killed on each necropsy day (Days 92 and 93).
All animals were weighed and examined externally. The abdominal cavity was opened and the animals were exsanguinated from the caudal vena cava. The cranial and thoracic cavities were opened and a full internal examination was performed. Any macroscopic abnormalities were recorded.


HISTOPATHOLOGY: Yes

For all animals, with the exception of the eyes, bone marrow smear, Harderian glands, optic nerves and testes, either whole organs or samples of the tissues listed below were preserved in neutral buffered formaldehyde. The eyes, Harderian glands and optic nerves were fixed in Davidson’s fluid and the testes were fixed in Modified Davidson’s. After fixation, all specified tissues were wax embedded. The bone marrow smears were fixed in methanol and then stained, but in the absence of any haematological reasons for doing so were not examined.

adrenal glands, ovaries, aorta, pancreas, bone marrow smear, pharynx, brain (3 levels examined), pituitary gland, prostate & seminal vesicles (including coagulating gland), caecum, colon, duodenum, rectum, epididymides, salivary gland (mandibular), eyes (including optic nerves), sciatic nerve, femur & femorotibial joint (including marrow), skeletal muscle, Harderian glands, spinal cord (3 levels examined LS and TS), heart, spleen, ileum, sternum with bone marrow, jejunum (including Peyer’s patch), stomach, kidneys (one LS and one TS), submandibular lymph nodes, lacrimal glands, testes, larynx, thymus, liver, thyroids (including parathyroids), lungs (including mainstem bronchi), trachea, mammary gland (collected with inguinal skin), urinary bladder, mesenteric lymph nodes, uterus and cervix, nasal cavity (one section – 3rd level), vagina, oesophagus, all gross lesions

All tissues from Control and high dose animals (in addition to gross lesions from all groups) were cut at a nominal thickness of 4 µm to 5 µm, stained with haematoxylin and eosin and examined microscopically. Subsequently (due to microscopic findings in the liver and kidneys of high dose animals), the liver and kidneys were also processed to slide and examined microscopically for intermediate and low dose animals.

Statistics:

All statistical tests were two-sided with minimum significance levels of 5 % and 1 %. Non-parametric statistics were not routinely conducted. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA). Data were examined for unusually high or low values which could influence the statistical analysis and interpretation (possible outliers). After examining for any outliers, if the variances were clearly heterogeneous, transformations (e.g. log, double arcsine or square root) was used in an attempt to stabilise the variances. If the transformations failed the data set was examined and a decision taken on further action.

For Quantitative Data: Body weight, cumulative body weight gain from the start of dosing, haematology, coagulation and blood chemistry parameters were analysed using a parametric ANOVA.
Organ weights were analysed using ANOVA for the absolute weights and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. Group summaries (mean, standard deviation and number of observations) and individual values are presented for organ weights as a percentage of body weight, but these were not evaluated for statistical significance.
Outliers: Exclusion of individual outlier values was considered unnecessary, as it was decided that the data reflected variation expected. The decision not to exclude values was considered not to have affected the scientific interpretation of the study findings.
Dunnett’s test: 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, and statistical flags are presented in the tables of results in the final report.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, Male 36 showed piloerection on Day 41 and Female 78 showed piloerection and unsteady gait on Day 5. Females 72 and 73 both had an abnormal gait for extended periods (Days 44 to 55 and Days 34 to 92, respectively).
Excessive salivation was seen after dosing on a number of occasions throughout the treatment period for both sexes given 25 or 75 mg/kg/day, but was considered to be non-adverse. There were no test item-related observations at 10 mg/kg/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall mean body weight gain was significantly reduced for males given 75 mg/kg/day (18 %, p<0.01), resulting in a significant reduction in their mean final body weight, relative to Controls (10 %, p<0.01,). Females given this dosage showed a notable, but transient, reduction in weight gain over Days 1 to 9, relative to Controls (33 %, p<0.01), largely attributable to Female 78, which lost 17 % body weight between Days 1 to 4. Thereafter, their weight gain was similar to that of Controls and their final body weight was within 3 % of group mean Control values.
There were no adverse effects on body weight at 10 or 25 mg/kg/day, where overall gains and final body weights were similar to those of the Controls.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no notable effect on food intake, as although mean values for test item-treated groups were reduced on various occasions during the study, overall mean food intake (Days 1 to 92) for all test item-treated groups was comparable with Controls.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Three females given 75 mg/kg/day had hyper reflectivity of the retina in one or both eyes in Week 12, which was considered to be test item-related as it was not seen for these animals before the start of treatment or in Controls. Subsequently, females given 25 mg/kg/day were also examined but no test item-related changes were observed. There were no test item related findings for males.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on haematology or coagulation parameters.
Where statistically significant differences were observed they were not consistent between the sexes (mean cell volume and relative reticulocytes) or lacked a strict dose relationship (red blood cell count, haemoglobin concentration, mean cell haemoglobin, red blood cell distribution width and platelets). Individual values were within or only marginally outside background ranges and therefore were considered to reflect normal biological variation rather than any effect of test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Group mean alanine aminotransferase (ALT) activity and total protein were statistically significantly higher (p<0.01) for males given 75 mg/kg/day when compared with Controls with most individual values being above the historical Control upper limit.
Bile acids were significantly (p<0.01) increased (+280 %) for females given 75 mg/kg/day and also increased (+94 %) for females given 25 mg/kg/day. These effects were not seen for males, there were no test item-related effects on associated parameters and there were no microscopic changes indicative of adverse effects on biliary function, this finding was considered not to be of toxicological significance.
Where other statistically significant differences were observed individual values within or only marginally outside background ranges and were therefore considered to reflect normal biological variation rather than any effect of the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

All females given 75 mg/kg/day were closely or loosely bunched in their cages on Days 21 and 35, respectively; five females at this dose level were also closely bunched in their cage on Day 28.
In the open field arena, three males given 75 mg/kg/day had roughened fur on Days 28 and 56. Abnormal gait (varying between slight and marked) was observed for females on various occasions during the study, most notably on Days 49, 56, 70 and 77, where six, five, seven and seven females were affected, respectively. A number of females at this dose level also had straub tail (four females on Day 49, five females on each of Days 70 and 77 and three females on Day 83). At 25 mg/kg/day, four and two males had roughened fur on Days 28 and 56, respectively; one male also had piloerection on Day 28.
Both sexes given 25 or 75 mg/kg/day were markedly more active than Controls for just the first 10 minutes of motor monitoring, with either no differences or no consistent sex difference from Controls for subsequent intervals. This increase in activity was considered to be test item-related.
Forelimb grip strength was significantly (p<0.01 to p<0.05) reduced for males given 25 or 75 mg/kg/day (-27 % and -22 %, respectively) with the majority of individual values within historical Control ranges (218.7 to 782.7). These changes were not dose-related, no similar effect was seen in the hindlimb grip strength and in the absence of any notable findings for females, this finding was considered not to be test item-related.
Where other observations were noted, these were either isolated or sporadic instances or were also seen for Controls and so were considered to be unrelated to administration of the test item.
There were no test item-related effects on functional observations at 10 mg/kg/day and no effects on sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli at any dose level.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean adjusted liver weights were higher than Controls for both sexes given 25 mg/kg/day (13 % and 14 % higher than Controls for males and females, respectively) or 75 mg/kg/day (41 % and 47 % higher than Controls for males and females, respectively). All female relative liver weights were within the Sequani background range. However, the relative liver weights for one male given 25 mg/kg/day and all 10 males given 75 mg/kg/day were above the Sequani background upper limits and therefore this increase was considered to be test item-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Three out of 10 males at 25 mg/kg/day and four out of 10 males at 75 mg/kg/day had mottled, abnormal coloured kidneys, which was not seen for Controls; one male at 75 mg/kg/day had right kidney pelvic dilatation. As the microscopic findings in these groups were considered to be unrelated, these macroscopic findings although considered to be test item-related were considered not to be adverse.
There were no test item-related macroscopic findings for males given 10 mg/kg/day or females at any dose level.
A variety of other spontaneous changes was noted in Control and treated animals with no indication of an effect of treatment. The spectrum of these findings was generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver:
Test item-related findings in the liver comprised of centrilobular vacuolation (at 10 (males only), 25 or 75 mg/kg/day) and centrilobular hypertrophy (at 25 (males only) or 75 mg/kg/day). The hepatic changes seen in animals given 25 or 75 mg/kg/day would account for the increased group mean adjusted liver weights in these animals. As a result of the changes in the centrilobular zone, there was an apparent compression of the periportal zone resulting in an atrophic appearance of the hepatocytes in this region.
The centrilobular vacuolation seen in males given 10 mg/kg/day was consistent with fatty macrovesicular change, which is often regarded as a non-adverse, physiological adaptation to an imbalance between uptake of lipids from the blood and secretion of lipoproteins by the hepatocyte. Furthermore, it was not associated with increased liver weight and, since this finding was graded mostly as minimal to slight, rather than the moderate to marked severity seen at higher doses, it was considered non-adverse at this dose level.

Kidney:
Tubular basophilia was observed in males at all dose levels, including one Control.
Although mottled kidneys (in three males given 25 mg/kg/day and four males given 75 mg/kg/day) were seen at necropsy, this was considered to be unrelated to the renal changes seen microscopically. Renal tubular basophilia is a common finding in male rats and although the incidence was highest in high dose males, there was no increase in severity at this dosage. Since the incidences at the low and intermediate dose levels did not show a trend with dose away from Controls, where one animal was also affected, this finding was considered not to be clear evidence of renal toxicity and so, potentially unrelated to administration of the test item.
A variety of other spontaneous changes was noted in Control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Histopathological findings: neoplastic:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Toxicokinetics:

In males, after repeat administration of the test item for 90 days, systemic exposure (AUC0-24) on Day 90 increased approximately proportionally with respect to dose.  However, in females, AUC0-24 increased more than proportionally with dose.  

Systemic exposure was higher in males at 10 mg/kg/day, but this ratio changed with increasing dose to be higher in females at the highest dose level of 75 mg/kg/day.  This suggested that in females the clearance of the test item may become saturated with increasing dose.

Applicant's summary and conclusion

Conclusions:

N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine administered once daily by oral gavage to the rat at doses of 0, 10, 25, or 75 mg/kg/day was tolerated for at least 90 days, but elicited adverse hepatic changes at 25 or 75 mg/kg/day. Hepatic changes seen for males given 10 mg/kg/day were considered to be non-adverse and in the absence of any other notable findings at this dose level, 10 mg/kg/day was concluded to represent the No Observed Adverse Effect Level (NOAEL) for both sexes.
After repeat administration of the substance for 90 days, systemic exposure (AUC0-24) on Day 90 increased approximately proportionally with respect to dose in males and more than proportionally in females. At the NOAEL, exposure was characterised by Cmax and AUC0-24 on Day 90 of 673 ng/mL and 3020 ng.h/mL for males and 372 ng/mL and 1650 ng.h/mL for females, respectively.

Executive summary:

The potential toxicity and toxicokinetics of N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine was assessed when administered orally, by gavage, to the rat once daily for at least 90 days.

Forty male and forty female rats of the Crl:WI(Han) strain were allocated to the study.  Groups of 10 males and 10 females were dosed with 0 (vehicle), 10, 25 or 75 mg/kg/day the test item once daily, by oral gavage, at a dose volume of 5 mL/kg for at least 90 days, until the day before necropsy.

Animals were examined daily from the start of treatment.  Body weights were recorded daily for the first two weeks of treatment and weekly thereafter and food intake was recorded daily for the first week of treatment and weekly thereafter, until necropsy.  The eyes of all animals were examined before the start of treatment, with Control and high dose animals also being examined in Week 12; due to findings seen for high dose females, intermediate dose females were also examined in Week 12.  Blood samples were taken for clinical pathology assessment during Week 13.  Blood samples were also taken on Day 90 at seven time-points for toxicokinetic evaluation.

Animals were subjected to standard arena observations pre-dose and once weekly thereafter.  Grip strength, motor activity and sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli were assessed once in Week 12.

All animals were subjected to a gross necropsy, specified organs were weighed and a full list of tissues was examined microscopically from the Control and high dose animals.  In addition, the liver and kidneys were examined microscopically from intermediate and low dose animals.

There were no deaths.  At 75 mg/kg/day, one female had an unsteady gait and piloerection on Day 5, one male showed piloerection on Day 41 and two females had an abnormal gait for extended periods (12 days and 59 days).  Excessive salivation was seen after dosing on a number of occasions throughout the treatment period for both sexes given 25 or 75 mg/kg/day.  There were no test item-related observations at 10 mg/kg/day.

Overall mean body weight gain and final body weight for males given 75 mg/kg/day were significantly reduced compared with Controls.  However, for females at this dose level, mean body weight gain and final body weight were only slightly reduced, relative to Controls.  There were no adverse effects on body weight at 10 or 25 mg/kg/day, where overall gains and final body weights were similar to those of the Controls.  There was no notable effect on food intake.

For females given 75 mg/kg/day, functional observations recorded on Days 21, 28 and 35 of dosing revealed slight changes in behaviour (closely or loosely grouped together) when group housed; abnormal gait and Straub tail were also observed in the arena for females at this dose level.  Both sexes given 25 or 75 mg/kg/day were markedly more active than Controls for just the first 10 minutes of motor monitoring, with either no differences or no consistent sex difference from Controls for subsequent intervals.  

Three females given 75 mg/kg/day had hyper reflectivity of the retina in one or both eyes in Week 12, which was considered to be test item-related as it was not seen for these animals before the start of treatment or in Controls.

Group mean alanine aminotransferase activity and total protein in males given 75 mg/kg/day and bile acids for females given 75 mg/kg/day were statistically significantly higher than in Controls.

In males, after repeat administration of the test item for 90 days, systemic exposure (AUC0-24) on Day 90 increased approximately proportionally with respect to dose.  However, in females, AUC0-24 increased more than proportionally with respect to dose.

Increased liver weights for animals given 25 or 75 mg/kg/day were associated with centrilobular vacuolation and hypertrophy (males only at 25 mg/kg/day).  Centrilobular vacuolation was also observed for males given 10 mg/kg/day but because it was graded as minimal to slight at this dosage and liver weight was unaffected it was deemed to be non- adverse.  Renal tubular basophilia was observed in males at all dose levels and although the incidence was highest in high dose males, the incidences at the low and intermediate dose levels were not strictly dose-related and the change was also observed in a Control male.