Registration Dossier

Administrative data

Description of key information

Short term repeated dose oral toxicity

Oral gavage administration of test item to Crl:CD(SD) rats for 28 consecutive days resulted in clinical signs (≥250 mg/kg/day), slightly higher food intake (≥500 mg/kg/day), slightly increased motor activity (males at ≥500 mg/kg/day) hematology changes (1000 mg/kg/day), urinalysis alterations (≥500 mg/kg/day), higher liver weights and correlating hepatocellular hypertrophy (1000 mg/kg/day) all of which were considered adaptive and nonadverse, necrosis of the nasal turbinates (most of the 1000 mg/kg/day group animals, some of the 500 mg/kg/day group males, and one 250 mg/kg/day group male) that was considered to be biologically irrelevant to this study, microscopic changes in reproductive organs (testicular germ cell degeneration and/or tubular degeneration and ovarian cysts) and altered estrous cycles (1000 mg/kg/day). The cause of the death of a 1000 mg/kg/day group female could not be determined and its association with the test substance was uncertain. The testicular tubular degeneration observed at 1000 mg/kg/day was considered to be potentially adverse. Based on these results, the no-observed-effect level (NOEL) was less than 250 mg/kg/day and the no-observed-adverse-effect level (NOAEL) was conservatively considered to be 500 mg/kg/day (based upon the uncertainty of the cause of death of one female and testicular tubular degeneration observed at 1000 mg/kg/day) (OECD 407).

Subchronic repeated dose oral toxicity

Oral gavage administration of test item to Crl:CD(SD) rats for a minimum of 91 consecutive days resulted in effects considered to be nonadverse. Specifically, clinical findings (≥250 mg/kg/day), statistically significantly lower body weights (males at 750 mg/kg/day and females at ≥500 mg/kg/day), statistically significantly increased motor activity (750 mg/kg/day), alterations (generally statistically significant) in hematology (750 mg/kg/day), serum chemistry (≥500 mg/kg/day), urinalysis (≥250 mg/kg/day), and organ weights (≥500 mg/kg/day), macroscopic findings (females at ≥500 mg/kg/day), and microscopic findings ≥250 mg/kg/day) were considered to be nonadverse. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 750 mg/kg/day (OECD 408).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 June 2014 to 11 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
various deviations with no impact on outcome of study (see below)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Crl:CD(SD) rats were used as the test system for this study. This species and strain of animal is recognized as appropriate for subchronic toxicity studies. The Sprague Dawley rat was selected because it is a widely used strain for which significant historical control data are available. The number of animals selected for this study was the minimum necessary to yield scientifically meaningful results based on the OECD Guideline for Testing of Chemicals (Guideline 407).
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION
- Crl:CD(SD) rats (25 males and 25 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 03-Jun-2014.
- The animals were approximately 42 days old at receipt.
- Each animal was examined by a qualified technician on the day of receipt and weighed on the following day.
- Each animal was uniquely identified with a subcutaneous microchip (BMDS system) implanted in the dorsoscapular area.
- All animals were housed for a minimum of a 17-day acclimation period. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior.
- Data collection during acclimation began on 04-Jun-2014. Individual body and food weights were recorded and detailed physical examinations were performed periodically during acclimation.

ANIMAL HOUSING
- Upon arrival, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week.
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research are accredited by AAALAC International.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research.
- Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinical pathology blood collection when food, but not water, was withheld.
- Municipal water supplying the facility was analyzed for contaminants according to SOPs. The results of the diet analyses are maintained in the study records and the results of the water analyses are maintained at WIL Research.
- No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- All animals were housed throughout acclimation and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71 ± 5°F (22 ± 3°C) and 50 ± 20%, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis.
- Actual mean daily temperature ranged from 70.5°F to 71.4°F (21.4°C to 21.9°C) and mean daily relative humidity ranged from 35.1% to 45.6% during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.
Route of administration:
oral: gavage
Details on route of administration:
ORGANISATION OF TEST GROUPS, DOSAGE/DOSE LEVELS AND TREATMENT REGIMEN
- The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible Teflon-shafted, stainless steel ball-tipped dosing cannula once daily for 28 consecutive days, through the day prior to the scheduled necropsy.
- The dose volume for all groups was 5 mL/kg.
- Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dosage. Adjusted doses became effective the day of collection of the weekly body weights.
- All animals were dosed at approximately the same time each day. The first day of dosing was study day 0; the first week of dosing was study week 0.
- Group assignment is shown in the table below.
- The dosage levels were determined from results of previous studies conducted orally in rats with similar test substances. It was anticipated that the high-dosage level would show drug-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation.
- The lower dosage levels were selected at intervals that were predicted to be narrow enough to reveal any dose-related trends.
- The selected route of administration for the study was oral (gavage).
Vehicle:
peanut oil
Remarks:
NF (lot no. 1CK0122, exp. date: 31-OCT-2014)
Details on oral exposure:
PREPARATION
- The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light. The vehicle was mixed throughout the sampling and dose administration procedures.
- Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSES
- Due to the nature of the test substance, stability was not determined in this study.
- Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 50 and 200 mg/mL dosing formulations. - Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared for the first and last weeks of dosing. All samples were shipped at room temperature, protected from light, to Wildlife International for analysis.
- All analyses were conducted by Wildlife International using a validated method.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
STUDY DESIGN
- An overview of study design is attached.

ASSIGNMENT OF ANIMALS TO TREATMENT GOUPS
- On 18-Jun-2014 (2 days prior to the initiation of dose administration), all available rats were weighed and examined in detail for physical abnormalities. These data were collected using WTDMs and reviewed by the Study Director.
- The animals judged suitable for assignment to the study were selected for use in a computerized
randomization procedure based on body weight stratification in a block design. A printout containing the animal numbers and individual group assignments was generated,
and the animals were then arranged into groups according to the printout.
- Individual body weights at randomization were within ± 20% of the mean for each sex.
- Animals not assigned to study were transferred to the WIL Research stock colony or euthanized by carbon dioxide inhalation and discarded.
- Each group (Groups 1-4) consisted of 5 males and 5 females.
- The animals were approximately 9 weeks old at the initiation of dose administration.
- Individual body weights ranged from 257 g to 293 g for males and from 189 g to 226 g for females at randomization.

DATA ACQUISITION AND REPORTING
- Data acquisition and reporting are summarised in the document attached.
Positive control:
Not applicable
Observations and examinations performed and frequency:
SURVIVAL
- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and their moribund state.
- The animal found dead was examined macroscopically as soon as possible to ensure that tissues were not lost due to autolysis.

CLINICAL OBSERVATIONS
- Clinical examinations were performed at the time of dose administration and approximately 1-2 hours following dose administration. The absence or presence of findings was recorded for individual animals at the scheduled intervals.
- Detailed physical examinations were conducted on all animals weekly (± 2 days) prior to randomization, on the day of randomization, weekly (± 2 days) during the study period, and on the day of the scheduled necropsy.

BODY WEIGHTS
- Individual body weights were recorded weekly (± 2 days) prior to randomization, on the day of randomization, on study day 0, weekly (± 2 days) during the study period, and on the day prior to the scheduled necropsy (nonfasted). Mean body weights and mean body weight changes were calculated for the corresponding intervals.
- Final body weights (fasted) were recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION
- Individual food weights were recorded weekly (± 2 days) prior to randomization, on the day of randomization, and weekly (± 2 days) throughout the study.
- Food consumption was calculated as g/animal/day for the corresponding body weight intervals.

FOB ASSESSMENTS
- FOB assessments were recorded for all surviving animals during study week 3.
- Testing was performed by the same technicians, whenever possible, without knowledge of the animals’ group assignments.
- The FOB was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB.
- The FOB used at WIL Research is based on previously developed protocols (Moser et al., 1991; Irwin, 1968; Gad, 1982; Moser et al., 1988; Haggerty, 1989; O'Donoghue, 1989).
- All animals were observed for the parameters listed in the table below.

LOCOMOTOR ACTIVITY
- Locomotor activity, recorded after the completion of the FOB, was assessed for all surviving animals during study week 3.
- Locomotor activity was measured automatically using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding a clear, plastic rectangular cage to quantify an animal’s locomotor activity.
- Four-sided black plastic enclosures were used to surround the clear plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by technicians or adjacent animals.
- The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams.
- The locomotor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB.
- The testing of treatment groups was conducted according to replicate sequence.
- Each animal was tested separately.
- Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six 10-minute subsessions for tabulation.
- Data for ambulatory and total locomotor activity were tabulated. Total locomotor activity was defined as a combination of fine locomotor skills (i.e., grooming; interruption of a single photobeam) and ambulatory locomotor activity (e.g., interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY
- Blood and urine samples for clinical pathology evaluations (hematology, coagulation, serum chemistry, thyroid hormones, and urinalysis) were collected from all surviving animals at the scheduled necropsy (study day 28).
- The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection.
- The animals were placed in a holding room in the WIL Research Necropsy Department for at least 1 hour prior to blood collection for hormone parameters to acclimate the animals and minimize stress.
- Blood was collected at the time of euthanasia via the inferior vena cava of animals anesthetized by isoflurane inhalation.
- Blood was collected into tubes containing potassium EDTA (hematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry and thyroid hormones).
- Hematology and coagulation parameters evaluated are shown in the table below.
- Serum chemistry parameters evaluated are shown in the table below.
- Urinalysis parameters evalulated are shown in the table below.
Sacrifice and pathology:
MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all animals.
- Animals were anesthetized by isoflurane inhalation and euthanized by exsanguination.
- The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The tissues and organs listed in the table below were collected and placed in 10% neutral-buffered formalin (except as noted).

ORGAN WEIGHTS
- Organs listed in the table below were weighed from all animals at the scheduled necropsy.
- Paired organs were weighed together.
- Organ to final body weight and organ to brain weight ratios were calculated.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION
- After fixation, protocol-specified tissues were trimmed according to WIL Research SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin.
- Microscopic examination was performed on all tissues listed from the animal that died in the 1000 mg/kg/day group and all animals in the control and 1000 mg/kg/day groups at the scheduled necropsy.
- Gross lesions were examined from all animals in the 250 and 500 mg/kg/day groups at the scheduled necropsy.
- In addition, the liver, thyroid gland, nasal cavity, testes, and female reproductive tissues (ovaries, uterus, cervix, and vagina) were identified as potential target tissues and were examined from all animals.
- Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.
Statistics:
STATISTICAL ANALYSIS
- Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean.
- Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.
- See below for full details of statistical analysis.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related clinical observations noted for the 500 and 1000 mg/kg/day group animals, generally in a dose-related manner, included the following: clear and/or yellow material around the mouth and yellow material on various body surfaces for males and females, and red material around the nose and/or mouth for females.
- In addition, clear discharge from the eyes was noted for females in the 1000 mg/kg/day group. These clinical findings were noted primarily at the time of dosing and/or 1-2 hours post-dosing
as early as study day 3 and generally persisted throughout the study.
- Test substance-related clinical observations noted for the 250 mg/kg/day group animals were limited to clear material around the mouth in males and females at the time of dosing and/or 1-2 hours post-dosing between study days 12 and 26.
- All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- One 1000 mg/kg/day group female (no. 9498) was found dead on study day 18.
- There were no clinical or gross observations for this animal and the cause of death was undetermined following microscopic evaluation and its association with the test substance was uncertain.
- All other animals survived to the scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Body weights were unaffected by test substance administration.
- There were no statistically significant differences when the control and test substance-treated groups were compared.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related slightly higher food consumption values were noted in the 500 and 1000 mg/kg/day group males and females.
- Food consumption in the 500 and 1000 mg/kg/day group males was slightly higher than the control group from study days 21 to 27.
- Food consumption in the 1000 mg/kg/day group females was higher than the control group from study days 7 to 14 and 14 to 21. In addition, food consumption in the 500 and 1000 mg/kg/day group females was slightly higher than the control group from study days 21 to 27.
- The higher food consumption values at 500 and 1000 mg/kg/day were considered test substance-related but not adverse due to the lack of statistical significance and absence of correlative body weight gains.
- There were no test substance-related effects on food consumption in the 250 mg/kg/day group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
HEMATOLOGY AND COAGULATION
- Higher percent and absolute reticulocyte values were noted in the 1000 mg/kg/day groups and lower platelet values were noted in the 1000 mg/kg/day group females.
- Higher absolute and percent reticulocyte values were noted in the 1000 mg/kg/day group males and females, and the differences were statistically significant. Almost all individual animal values were above the range of the control group. There were no corresponding alterations in erythrocyte count values.
- Lower platelet values were noted in the 1000 mg/kg/day group females and the difference was statistically significant. All individual animal values were lower than the range of the control group and some were also lower than the range of the WIL Research historical control data.
- There were no other test substance-related effects on hematology or coagulation parameters. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Lower white blood cell count and absolute lymphocyte count values were noted in the test substance-treated female groups. A dose response was lacking, all individual animal values were within the WIL Research historical control data range, and some of the control group values were at the high end of the WIL Research historical control data range; therefore, the differences were not considered to be test substance-related.
- Lower absolute basophil count values were noted in the 250 and 1000 mg/kg/day group females. A dose response was lacking and the low end of the WIL Research historical control data range was 0; therefore, the change was not attributed to test substance administration.
- A higher absolute monocyte count was noted in the 1000 mg/kg/day group females. All individual animal values were within the WIL Research historical control data range and the direction of the change was opposite that of the other leukocyte types and overall leukocyte count; therefore, the difference was not considered to be test substance-related.
- Statistically significant findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
SERUM CHEMISTRY
- There were no test substance-related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test substance-treated groups were compared.
- Lower total bilirubin values were noted in the 250, 500, and 1000 mg/kg/day group females. The low end of the WIL Research historical control data range is 0; therefore, the direction of change was not considered toxicologically relevant and the difference was not attributed to test substance administration.
THYROID HORMONES
- Statistically significantly lower total triiodothyronine (T3) values were noted in the 500 and 1000 mg/kg/day group males, and lower T3 values were also noted in the 250 mg/kg/day group males and the 1000 mg/kg/day group females. However, a definite dose response was lacking and there were no accompanying trends in thyroid stimulating hormone (TSH) values.
- TSH was slightly higher in the 1000 mg/kg/day group males and females but the differences were not significant and the TSH values for the 250 and 500 mg/kg/day group males and the 500 mg/kg/day group females were lower than that of the control group.
- A dose-related trend of lower thyroxine (T4) values was noted for males but not for females, but the difference was not statistically significant. Therefore, the association of the lower T3 values with test substance administration was uncertain.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Lower specific gravity and higher total volume values were noted in the 500 mg/kg/day group males and the 1000 mg/kg/day group males and females. All of the differences were statistically significant except for the difference in specific gravity for the 1000 mg/kg/day group females, and most individual animal values were outside of the range of the control group.
- The lower specific gravity was considered to be a byproduct of the higher total volume. There were no corresponding microscopic observations in the urinary tract.
- There were no other test substance-related effects on urinalysis parameters. However, a statistically significantly lower pH value was noted for the 500 mg/kg/day group females. A dose response was lacking and this difference was not attributed to the test substance.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
HOME CAGE OBSERVATIONS
- Home cage observations were unaffected by test substance administration.
- There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
HANDLING OBSERVATIONS
- Increased incidences of slightly soiled fur were noted in the 500 and 1000 mg/kg/day group males and females at the study week 3 evaluation when compared to the control group (statistically significant for the 1000 mg/kg/day group females).
- These findings correlated with the clinical observations of yellow material on various body surfaces noted in the same groups and were therefore considered test substance-related, but not adverse.
- There were no other statistically significant differences in handling observations when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
OPEN FIELD OBSERVATIONS
- Open field observations were unaffected by test substance administration.
- There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
SENSORY OBSERVATIONS
- Sensory observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
NEUROMUSCULAR OBSERVATIONS
- Neuromuscular observations were unaffected by test substance administration.
- Statistically significantly lower mean rotarod performance times were noted for the 250 and 500 mg/kg/day group males at the study week 3 evaluation. However, similar differences in rotarod performance were not observed in the 1000 mg/kg/day group males or in the females at any dose level; therefore, the differences were attributed to normal biological variation and not to test substance administration.
- There were no other statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
PHYSIOLOGICAL OBSERVATIONS
- Physiological observations were unaffected by test substance administration.
- Mean body temperature in the 1000 mg/kg/day females was statistically significantly lower than the control group at the study week 3 evaluation. However, the difference was slight (-0.9°C) and was considered to represent normal biological variation rather than an effect of test substance administration.
- There were no other statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Higher liver weights and ovaries/oviducts weights were noted in the 1000 mg/kg/day groups.
- Higher liver weights (absolute and relative to body and brain weights) were noted in the 1000 mg/kg/day group males and females and the differences were statistically significant. All individual animal values were higher than the range of the control group and many were also higher than the range of the WIL Research historical control data. This correlated with diffuse hepatocellular hypertrophy microscopically.
- Higher ovaries/oviducts weights (absolute and relative to body and brain weights) were noted in the 1000 mg/kg/day group females and the difference in ovaries/oviducts weight relative to body weight was statistically significant. Almost all individual animal values were higher than the range of the control group and there was a higher incidence of ovarian cysts observed microscopically in the 1000 mg/kg/day group.
- Statistically significantly lower thymus weights (absolute and relative to brain weights) were noted in the 1000 mg/kg/day group females. Lower thymus weights were also noted in the 1000 mg/kg/day group males and the 250 and 500 mg/kg/day group females, although the differences were not statistically significant. There was no corresponding lymphoid depletion microscopically and most individual animal weights were within the range of the WIL Research historical control data; therefore, the association with test substance administration was uncertain.
- There were no other test substance-related effects on organ weights. However, some statistically significant differences were observed when the control and test substance treated groups were compared.
- Higher kidney weight relative to body weight was noted in the 1000 mg/kg/day group females. This group had a lower final body weight and several control group animals had individual values that were lower than the range of the WIL Research historical control data. Therefore, the difference was not considered to be related to test substance administration.
- Lower seminal vesicles/prostate weight relative to body and brain weight values were noted in the 500 mg/kg/day group males. A dose response was lacking and several control group animals had individual values that were higher than the WIL Research historical control data range; therefore, the differences were not attributed to the test substance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no test substance-related macroscopic findings at the scheduled necropsy.
- All macroscopic findings noted were considered to be spontaneous and/or incidental in nature and unrelated to test substance administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic findings were noted in the liver, thyroid gland, nasal cavity, and reproductive tissues (see Table 3, attached).
- Minimal to mild hepatocellular hypertrophy was observed in most of the 1000 mg/kg/day group animals and 2/5 of the 500 mg/kg/day group males. The change was diffuse (lacked a lobular distribution). Hepatocytes were uniformly enlarged with abundant eosinophilic cytoplasm.
- Minimal to mild follicular cell hypertrophy and decreased colloid were observed in the thyroid glands of several of the 500 and 1000 mg/kg/day group males and females. Minimal follicular cell hypertrophy and decreased colloid were also observed in a single 250 mg/kg/day group male (animal no. 9455). Follicular cell hypertrophy was characterized by the presence of tall cuboidal to columnar follicular cells with increased amounts of basophilic, vacuolated cytoplasm. It was accompanied by decreased amounts of colloid throughout the thyroid glands in most of the animals.
- Minimal germ cell degeneration and/or tubular degeneration were observed in the testes of the 500 and 1000 mg/kg/day group males as well as a single 250 mg/kg/day group male (animal no. 9455). Germ cell degeneration was characterized by single cell necrosis of pachytene spermatocytes in the stage XIV tubules. Affected cells were shrunken, rounded, and hypereosinophilic, with pyknotic nuclei. Tubular degeneration was observed in one 1000 mg/kg/day group male and was characterized by scattered seminiferous tubules exhibiting diffuse loss of germ cells and spermatids and vacuolation of remaining Sertoli cells.
- A higher incidence of ovarian cysts was observed in the 1000 mg/kg/day group females when compared with the control group. A single follicular cyst was observed in 1 control group animal (no. 9497) and multiple follicular cysts were observed in one 250 mg/kg/day group animal (no. 9485), and a low incidence of ovarian cysts can be observed as a spontaneous background finding in rats (WIL Research historical control data, version 3.3). However, multiple follicular cysts were observed in the ovaries of 2 of the 1000 mg/kg/day group females and a luteal cyst was present in 1 female (no. 9482). In addition, all of the 1000 mg/kg/day group females were in diestrus, whereas at least 1 female each from the other groups was in proestrus or estrus. Given the test substance-related findings in the testes of the 1000 mg/kg/day group males, these changes in females may also have been test substance-related.
- Necrosis of the nasal turbinates was observed in the 1000 mg/kg/day group males and females and the 500 mg/kg/day group males, particularly within the posterior nasal cavity sections (nasal levels III and IV). Minimal necrosis was also observed in the nasal cavity of a single 250 mg/kg/day group male (animal no. 9455). Necrosis was characterized by degeneration and loss of olfactory and respiratory epithelial cells, erosion of the mucosa, and acute inflammation. Remaining epithelial cells within affected areas were flattened and jumbled and exhibited basophilic cytoplasm and large vesicular nuclei, interpreted as attempts at regeneration. There was no specific distribution pattern aside from the posterior sections being most severely affected. The change was multifocal to coalescing within these sections and affected both olfactory and respiratory epithelial cells. There were no test substance-related histologic changes within other respiratory system tissues.
- Minimal single cell necrosis was present in the liver of several of the 500 and 1000 mg/kg/day group males and females. This change was characterized by shrunken, rounded hepatocytes with hypereosinophilic cytoplasm and pyknotic nuclei. Although the change was observed at a higher incidence in the test substance-treated animals, it was also observed in a single control group male, was of minimal severity, is a common spontaneous background finding in the liver of rats (WIL Research historical control data, version 3.3), and was often located adjacent to mononuclear infiltrates in both the control and test substance-treated animals; therefore, it was not considered likely to be due to test substance administration.
- There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
LOCOMOTOR ACTIVITY
- Mean overall cumulative ambulatory activity counts for the 500 and 1000 mg/kg/day group males were statistically significantly higher (66.7% and 71.2%, respectively) than the control group values.
- In addition, the mean values for the 0-10 and 21-30 minute epochs in the 1000 mg/kg/day group males were statistically significantly higher than the control group.
- However, mean cumulative total activity counts for the 500 and 1000 mg/kg/day group males were only 44.8% and 23.5% higher, respectively, than the control group (not statistically significant), and activity counts (total and ambulatory) for females in these groups were generally lower than the control group values. Therefore, while apparently test substance-related, the slightly increased ambulatory activity noted in males at 500 and 1000 mg/kg/day was not considered adverse.
- There were no other statistically significant changes for the test substance-treated males and females when compared to the control group at the study week 3 evaluation. Values obtained from the 6 epochs evaluated (0-10 minutes, 11-20 minutes, 21-30 minutes, 31-40 minutes, 41-50 minutes, and 51-60 minutes) and the overall 60-minute test session were comparable to the concurrent control values.
- No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated on study week 3.
Dose descriptor:
NOEL
Effect level:
< 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
histopathology: non-neoplastic
Critical effects observed:
no

ANALYSIS OF DOSING FORMULATIONS

- With the exception of a mean percent recovery of 60.3% in the low dose (50 mg/mL) formulations following resuspension sampling during week 0, all formulation sample mean percent recoveries were between approximately 75% and 94%. The results of the analyses were consistent with results obtained during the method validation 70-day storage assessment in that formulations at lower concentrations have lower mean percent recovery following longer storage duration.

- For the samples in this study, the number of days from preparation/sampling of the formulations to time of analyses was 41 to 62 days. However, all formulations were used for dosing within 8 days of preparation. Therefore, it is believed that mean percent recoveries of the study samples would be increased if they had been analyzed closer to the time of sampling and that animals on this study received the appropriate dose concentration/dosage levels during this study.

- Results of homogeneity and concentration analyses of dosing formulations are summarized in the Table 1 and Table 2 (attached).

Conclusions:
Oral gavage administration of test item to Crl:CD(SD) rats for 28 consecutive days resulted in clinical signs (≥250 mg/kg/day), slightly higher food intake (≥500 mg/kg/day), slightly increased motor activity (males at ≥500 mg/kg/day) hematology changes (1000 mg/kg/day), urinalysis alterations (≥500 mg/kg/day), higher liver weights and correlating hepatocellular hypertrophy (1000 mg/kg/day) all of which were considered adaptive and nonadverse, necrosis of the nasal turbinates (most of the 1000 mg/kg/day group animals, some of the 500 mg/kg/day group males, and one 250 mg/kg/day group male) that was considered to be biologically irrelevant to this study, microscopic changes in reproductive organs (testicular germ cell degeneration and/or tubular degeneration and ovarian cysts) and altered estrous cycles (1000 mg/kg/day). The cause of the death of a 1000 mg/kg/day group female could not be determined and its association with the test substance was uncertain. The testicular tubular degeneration observed at 1000 mg/kg/day was considered to be potentially adverse. Based on these results, the no-observed-effect level (NOEL) was less than 250 mg/kg/day and the no-observed-adverse-effect level (NOAEL) was conservatively considered to be 500 mg/kg/day (based upon the uncertainty of the cause of death of one female and testicular tubular degeneration observed at 1000 mg/kg/day).
Executive summary:

GUIDELINE

The objective of this study was to evaluate the potential toxic effects of the test substance when administered via gavage to rats for 28 consecutive days according to the OECD Guideline for Testing of Chemicals (Guideline 407). This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment.

 

METHODS

The test substance in the vehicle (peanut oil) was administered orally by gavage once daily for 28 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 250, 500, and 1000 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Each group consisted of 5 animals/sex. Following 28 days of dose administration, all surviving rats were euthanized. All animals were observed twice daily for mortality and their moribund state. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body and food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and locomotor activity data were recorded for all surviving animals during study week 3. Clinical pathology parameters (hematology, coagulation, serum chemistry, thyroid hormones, and urinalysis) were analyzed for all surviving rats at the scheduled necropsy (study day 28). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from the animal that died and all animals in the control, 250 (gross lesions only), 500 (gross lesions only), and 1000 mg/kg/day groups at the scheduled necropsy. In addition, the liver, thyroid gland, nasal cavity, testes, and female reproductive tissues (ovaries, uterus, cervix, and vagina) were identified as potential target tissues and were examined from all animals.

 

RESULTS

One 1000 mg/kg/day group female was found dead on study day 18. As there were no clinical or macroscopic findings for this animal, the cause of death was undetermined and its association with the test substance was uncertain. All other animals survived to the scheduled necropsy. There were no test substance-related effects on body weight, alterations in serum chemistry parameters, or macroscopic findings.

Test substance-related clinical observations were noted in the 500 and 1000 mg/kg/day group males and females, generally in a dose-related manner, including clear and/or yellow material around the mouth, yellow material on various body surfaces, and red material around the nose and/or mouth for females. In addition, clear discharge from the eyes was noted for females in the 1000 mg/kg/day group. These clinical findings were noted primarily at the time of dosing and/or 1-2 hours post-dosing as early as study day 3 and generally persisted throughout the study.

Test substance-related clinical observations in the 250 mg/kg/day group included clear material around the mouth in males and females at the time of dosing and/or 1-2 hours post-dosing between study days 12 and 26.

Test substance-related increased incidences of slightly soiled fur were noted for males and females in the 500 and 1000 mg/kg/day groups at the study week 3 functional observational battery evaluation. In addition, test item-related, slightly increased ambulatory activity was noted in males at 500 and 1000 mg/kg/day at the study week 3 locomotor activity assessment.

Test substance-related slightly higher food consumption values were noted in the 500 and 1000 mg/kg/day group males from study days 21 to 27, the 500 mg/kg/day group females from study days 21 to 27, and the 1000 mg/kg/day group females from study days 7 to 27. The higher food consumption values in these groups were considered test substance-related but not adverse due to the absence of correlative body weight gains.

Notable clinical pathology alterations for animals administered 1000 mg/kg/day included higher reticulocytes (males and females), lower platelets (females), lower urine specific gravity (males and females), and higher urine total volume (males and females). Lower urine specific gravity and higher urine total volume was also noted in males at 500 mg/kg/day. There were no correlating microscopic findings.

Lower triiodothyronine (T3) values were noted in the 500 and 1000 mg/kg/day group males; however, there were no corresponding alterations in thyroid stimulating hormone (TSH) and therefore the toxicologic relevance and association of this change with the test substance was uncertain.

Minimal to mild hepatocellular hypertrophy was observed in most of the 1000 mg/kg/day group animals and 2/5 of the 500 mg/kg/day group males, corresponding to higher liver weights noted for 1000 mg/kg/day group animals.. There were no accompanying alterations in clinical pathology or test substance-related microscopic findings indicative of hepatotoxicity; therefore, the change was considered to be nonadverse.

Minimal to mild follicular cell hypertrophy and decreased colloid were observed in the thyroid glands of several of the 500 and 1000 mg/kg/day group males and females and a single 250 mg/kg/day group male. The thyroid changes in these animals were considered to be secondary to enzyme induction rather than evidence of direct thyroid toxicity. Minimal germ cell degeneration and/or tubular degeneration were observed in the testes of the 500 and 1000 mg/kg/day group males as well as a single 250 mg/kg/day group male. A higher incidence of ovarian cysts was observed in the 1000 mg/kg/day group females when compared with the control group, corresponding to the higher ovaries/oviducts weights for these females. The cause of these effects is uncertain. They may reflect a direct toxicity of the test substance on the reproductive tract or may have been secondary to the changes observed in the thyroid gland.

Necrosis of the nasal turbinates was observed in the 1000 mg/kg/day group males and females and the 500 mg/kg/day group males, particularly within the posterior nasal cavity sections (nasal levels III and IV). Minimal necrosis was also observed in the nasal cavity of a single 250 mg/kg/day group male. This nonspecific extensive necrosis and inflammation of the posterior nasal cavity was consistent with gavage-related reflux of the test substance.

Lower thymus weights were also noted in the 250 and 500 mg/kg/day females and 1000 mg/kg/day group males and females. There was no corresponding lymphoid depletion microscopically and most individual animal weights were within the range of the WIL Research historical control data; therefore, the association with test substance administration was uncertain.

 

CONCLUSIONS

Oral gavage administration of test item to Crl:CD(SD) rats for 28 consecutive days resulted in clinical signs (≥250 mg/kg/day), slightly higher food intake (≥500 mg/kg/day), slightly increased motor activity (males at ≥500 mg/kg/day) hematology changes (1000 mg/kg/day), urinalysis alterations (≥500 mg/kg/day), higher liver weights and correlating hepatocellular hypertrophy (1000 mg/kg/day) all of which were considered adaptive and nonadverse, necrosis of the nasal turbinates (most of the 1000 mg/kg/day group animals, some of the 500 mg/kg/day group males, and one 250 mg/kg/day group male) that was considered to be biologically irrelevant to this study, microscopic changes in reproductive organs (testicular germ cell degeneration and/or tubular degeneration and ovarian cysts) and altered estrous cycles (1000 mg/kg/day). The cause of the death of a 1000 mg/kg/day group female could not be determined and its association with the test substance was uncertain. The testicular tubular degeneration observed at 1000 mg/kg/day was considered to be potentially adverse. Based on these results, the no-observed-effect level (NOEL) was less than 250 mg/kg/day and the no-observed-adverse-effect level (NOAEL) was conservatively considered to be 500 mg/kg/day (based upon the uncertainty of the cause of death of one female and testicular tubular degeneration observed at 1000 mg/kg/day).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2014 to 27 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
various deviations with no impact on outcome of the study (see Appendix A, attached)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Crl:CD(SD) rats were used as the test system for this study. This species and strain of animal is recognized as appropriate for subchronic oral (gavage) toxicity studies. The Sprague Dawley rat was selected because it is a widely used strain for which significant historical control data are available. The number of animals selected for this study was the minimum necessary to yield scientifically meaningful results based on the OECD Guideline for Testing of Chemicals: Guideline 408.
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION
- Crl:CD(SD) rats (55 males and 55 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 22-Jul-2014. The animals were approximately 5 weeks old at receipt.
- Each animal was examined by a qualified technician on the day of receipt and weighed 3 days later.
- Each animal was uniquely identified with a subcutaneous microchip (BMDS system) implanted in the dorsoscapular area.
- All animals were housed for a 14-day acclimation period. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior.
- Data collection during acclimation began on 25-Jul-2014. Individual body and food weights were recorded and detailed physical examinations were performed periodically during acclimation. In addition, ophthalmic examination data were recorded for animals during acclimation.

ANIMAL HOUSING
- Upon arrival and throughout the study, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week.
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research are accredited by AAALAC International.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research.
- Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinical pathology blood collection and the scheduled necropsies when food, but not water, was withheld.
- Municipal water supplying the facility was analyzed for contaminants according to the WIL Research SOPs. The results of the diet and water analyses are maintained at WIL Research.
- No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.
- In order to maintain the health status of individual animals during the study, water bottles were offered to animals as indicated in the attached table.

ENVIRONMENTAL CONDITIONS
- All animals were housed throughout acclimation and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71 ± 5°F (22 ± 3°C) and 50 ± 20%, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 70.6°F to 72.5°F (21.4°C to 22.5°C) and mean daily relative humidity ranged from 34.1% to 70.0% during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.
Route of administration:
oral: gavage
Details on route of administration:
ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible Teflon-shafted, stainless steel ball-tipped dosing cannula once daily for 91-92 consecutive days, through the day prior to the primary necropsy.
- The dose volume for all groups was 5 mL/kg. Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dosage. Adjusted doses became effective the day of collection of the weekly body weights.
- All animals were dosed at approximately the same time each day. The first day of dosing was study day 0; the first week of dosing was study week 0.
- Group assignment is shown in the table below.
- Dosage levels were selected based on results from a previous study (Haas, 2015, WIL-168211) conducted orally by gavage in rats with the test item. It was anticipated that the high-dosage level would show test substance-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation.
- The lower dosage levels were selected at intervals that were predicted to be narrow enough to reveal any dose-related trends.
- The selected route of administration for this study was oral (gavage).
Vehicle:
peanut oil
Remarks:
NF (lot nos. 1DB0384, 2DF0016, and 2DI0019, exp. dates: 31-Jan-2015, 30-Apr-2015, and 31-Aug-2015, respectively)
Details on oral exposure:
PREPARATION
- The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light.
- The vehicle was mixed throughout the sampling and dose administration procedures.
- Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light.
- The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSES
- Due to the hydrolytic nature of the test substance, stability was not determined on this study.
- Samples for homogeneity determination were collected from the top, middle, and bottom strata of the first 50 and 150 mg/mL dosing formulations prepared for administration to Groups 2 and 4.
- In addition, aliquots of sufficient volume for dosing a group of animals for 1 day were prepared and stored at room temperature, protected from light, for 8 days. The representative aliquots were then mixed with a magnetic stirrer for a minimum of 30 minutes, and samples were collected from the top and bottom strata of each aliquot for resuspension homogeneity determinations.
- Samples for concentration analysis were collected from the middle stratum of the study week 0 (homogeneity samples from the 50 and 150 mg/mL dosing formulations were used), 3, 7, and 12 dosing formulations (including the control group). All samples were shipped at room temperature, protected from light, to Wildlife International, Easton, MD, for analysis. All analyses were conducted by Wildlife International using a validated method (Wildlife International Project No.: 769C-102).
Duration of treatment / exposure:
91 or 92 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- Control and 750 mg/kg/day groups: 15 males and 15 females
- 250 mg/kg/day and 500 mg/kg/day groups: 10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
STUDY DESIGN
- An overview of study design is attached.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- On 01-Aug-2014 (4 days prior to the initiation of dose administration), all available rats were weighed and examined in detail for physical abnormalities. These data were collected using WTDMS and reviewed by the Study Director.
- The animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design. A printout containing the animal numbers and individual group assignments was generated, and the animals were then arranged into groups according to the printout.
- Individual body weights at randomization were within ± 20% of the mean for each sex. Animals not assigned to study were transferred to the WIL Research stock colony or euthanized by carbon dioxide inhalation.
- The control and 750 mg/kg/day groups (Groups 1 and 4, respectively) each consisted of 15 males and 15 females, and the 250 and 500 mg/kg/day groups (Groups 2 and 3, respectively) each consisted of 10 males and 10 females (10 animals/sex/group were euthanized following a minimum of 91 days of dose administration; the remaining 5 animals/sex in Groups 1 and 4 were euthanized following a minimum 28-day nondosing [recovery] period). These animals were then randomized into 5 study replicates to allow for the reasonable conduct of the functional observational battery and locomotor activity assessments. Each dose group and sex was approximately equally represented within each study replicate.
- The animals were approximately 7 weeks old at the initiation of dose administration.
- Individual body weights ranged from 185 g to 234 g for males and from 151 g to 185 g for females at randomization.

DATA ACQUISITION AND REPORTING
- Data acquisition and reporting are summarised in the document attached.
Positive control:
Not applicable
Observations and examinations performed and frequency:
SURVIVAL
- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

CLINICAL OBSERVATIONS
- Clinical examinations were performed at the time of dose administration and 1-2 hours following dose administration.
- During the recovery period, the animals were observed once daily.
- The absence or presence of findings was recorded for individual animals at the scheduled intervals.
- Detailed physical examinations were conducted on all animals 1 week (± 2 days) prior to randomization, on the day of randomization, weekly (± 2 days) during the study period, and on the days of the scheduled necropsies.
- Any observations noted outside of the above-specified intervals were also recorded.

BODY WEIGHTS
- Individual body weights were recorded 1 week (± 2 days) prior to randomization, on the day of randomization, on study day 0 (prior to dosing), weekly (± 2 days) during the study period, and on the day prior to the first day of the scheduled necropsies (nonfasted).
- The last nonfasted body weights were collected on the final days of study weeks 12 and 16, which were the days prior to each animal’s scheduled necropsy. These second weekly body weights during study weeks 12 and 16 are identified in the text and on the report tables as study weeks 13 and 17, respectively.
- Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the days of the scheduled necropsies.

FOOD CONSUMPTION
- Individual food weights were recorded 1 week (± 2 days) prior to randomization, on the day of randomization, and weekly (± 2 days) throughout the study period.
- Food consumption was calculated as g/animal/day for the corresponding body weight intervals.
- The last food consumption data were collected on the final days of study weeks 12 and 16, which were the days prior to each animal’s scheduled necropsy. These second weekly food consumption data during study weeks 12 and 16 are identified in the text and on the report tables as study weeks 13 and 17, respectively.
- When food consumption could not be measured for a given interval (due to spillage, weighing error, obvious
erroneous value, etc.), the appropriate interval was footnoted as "NA" on the individual tables.

FOB ASSESSMENTS
- FOB assessments were recorded for all animals during the final week of dose administration (study week 12/13). - Testing was performed by the same technicians, whenever possible, without knowledge of the animals’ group assignments.
- The FOB was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB.
- The FOB used at WIL Research is based on previously developed protocols (Moser et al., 1991; Irwin, 1968; Gad, 1982; Moser et al., 1988; Haggerty, 1989; O'Donoghue, 1989).
- All animals were observed for the parameters described in the table below.

LOCOMOTOR ACTIVITY
- Locomotor activity, recorded after the completion of the FOB, was assessed for all animals during the final week of dose administration (study week 12/13).
- Locomotor activity was measured automatically using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify an animal’s locomotor activity.
- Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams.
- The locomotor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. The testing of treatment groups was conducted according to replicate sequence.
- Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session
duration was 60 minutes. These data were compiled as six 10-minute subsessions for tabulation.
- Data for ambulatory and total locomotor activity were tabulated. Total locomotor activity was defined as a combination of fine locomotor skills (i.e., grooming; interruption of a single photobeam) and ambulatory locomotor activity (e.g., interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY
- Blood and urine samples for clinical pathology evaluations (hematology, coagulation, serum chemistry, and urinalysis) were collected at the scheduled necropsies (study weeks 13 and 17) for animals scheduled for necropsy. - The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection.
- Blood was collected at the time of euthanasia via the inferior vena cava of animals anesthetized by inhalation of isoflurane. Blood was collected into tubes containing potassium EDTA (hematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).
- Hematology and coagulation parameters evaluated are shown in the table below.
- Serum chemistry parameters evaluated are shown in the table below.
- Urinalysis parameters evalulated are shown in the table below.

OPTHALMIC EXAMINATIONS
- Ocular examinations were conducted on all animals during acclimation (27-Jul-2014; study week -2), during the final week of the dosing period (01-Nov-2014; study week 12), and during the final week of the recovery period (30-Nov-2014; study week 16).
- All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.
Sacrifice and pathology:
MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all animals. Animals were euthanized by inhalation of isoflurane followed by exsanguination.
- The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
- Clinical findings that were confirmed macroscopically were designated as correlates with externally observed (CEO) on the individual macroscopic data tables.
- The tissues and organs listed in the table below were collected and placed in 10% neutral-buffered formalin (except as noted).

ORGAN WEIGHTS
- Organs listed in the table below were weighed from all animals at the scheduled necropsies.
- Paired organs were weighed together. Organ to final body weight and organ to brain weight ratios were calculated.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION
- After fixation, protocol-specified tissues were shipped to Vet Path Services, Inc., Mason, OH, for processing.
- The tissues were trimmed according to the Vet Path Services, Inc. SOPs and the protocol.
- Trimmed tissues were processed into paraffin blocks and sectioned, and all tissues from animals in the control and 750 mg/kg/day groups at the primary necropsy and all gross lesions were mounted on glass microscope slides and stained with hematoxylin and eosin.
- In addition, slides were prepared for the following target tissues (i) Liver and thyroid glands from the 250 and 500 mg/kg/day group males and females at the primary necropsy and from the control and 750 mg/kg/day group males and females at the recovery necropsy (ii) Ovaries from the 250 and 500 mg/kg/day group females at the primary necropsy and the control and 750 mg/kg/day group females at the recovery necropsy.
- Microscopic examination was performed on all tissues listed from all animals in the control and 750 mg/kg/day groups at the primary necropsy and on all gross lesions at the primary and recovery necropsies.
- In addition, the target tissues listed above were examined microscopically.
- Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate.
- Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.
Statistics:
STATISTICAL ANALYSIS
- Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean.
- Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly.
- Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.
- See below for full details of statistical analysis.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related clinical observations were noted for the 250, 500, and 750 mg/kg/day group males and females during the dosing period.
- Test substance-related increased incidences of clear and yellow material around the mouth and yellow material around the anogenital, urogenital, and/or facial areas and on the forelimb(s), hindlimb(s), ventral neck, and/or ventral trunk were noted for the 250, 500, and 750 mg/kg/day group males and females during the dosing period when compared to the control group. In addition, test substance-related red material around the mouth was noted for the 250, 500, and 750 mg/kg/day group males and the 500 and 750 mg/kg/day group females.
- These clinical findings were primarily noted at the time of dosing and/or 1-2 hours post-dosing and generally persisted throughout the dosing period. None of these observations persisted to the recovery period, with the exception of 3 males in the 750 mg/kg/day group that were noted with yellow material around the anogenital and/or urogenital areas early in the recovery period (through study day 97).
- All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
- All animals survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related effects on body weights were noted for the 750 mg/kg/day group males and the 500 and 750 mg/kg/day group females during the dosing period.
- Males in the 750 mg/kg/day group were noted with mean body weight losses or lower mean body weight gains beginning during study week 4-5 and continuing through study week 11-12 when compared to the control group; the differences were statistically significant during study weeks 4-5 and 11-12. In addition, statistically significantly lower mean cumulative body weight gains were noted for the 750 mg/kg/day group males beginning at study week 11. As a result of these changes, statistically significantly lower mean body weights were noted for the 750 mg/kg/day group males at study weeks 11-13, with body weights 11.2% lower than the control group at study week 13.
- Mean body weight gains in the 750 mg/kg/day group were similar to the control group during the recovery period; however, the lower mean body weight in this group persisted through the recovery necropsy.
- Females in the 500 and 750 mg/kg/day groups were noted with mean body weight losses or slightly lower mean body weight gains sporadically during the dosing period, beginning during study week 2-3 compared to the control group.
- Statistically significantly lower mean cumulative body weight gains were noted for the 500 and 750 mg/kg/day group females beginning at study week 9 and 8, respectively, and generally continued through the end of the dosing period. As a result of these changes, statistically significantly lower mean body weights were noted for the 500 and 750 mg/kg/day group females at study weeks 11 and 12, with mean body weights 5.7% and 5.0% lower than the control group, respectively, at study week 13.
- Mean body weight gains for the 750 mg/kg/day group were similar to the control group during the recovery period; however, the lower mean body weight in this group persisted through the recovery necropsy.
- None of these changes in body weights were considered to be adverse.
- There were no other test substance-related effects on body weight.
- Additional differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Food consumption was unaffected by test substance administration.
- Differences from the control group were slight, not statistically significant, and/or the changes did not occur in a dose-related manner, with the following exceptions.
- Statistically significantly higher mean food consumption values were noted for the 750 mg/kg/day group females during study weeks 1-2, 5-6, and 11-12 and for the 250 mg/kg/day group females during study week 11-12 when compared to the control group. However, these changes were transient and/or occurred in a direction opposite of that noted for body weights. Therefore, these changes were not considered test substance-related.
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 ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups.
- All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
HEMATOLOGY AND COAGULATION
- There were test substance-related alterations in leukocyte, erythrocyte, and coagulation parameters noted primarily in the 750 mg/kg/day group animals at the primary necropsy.
- Leukocyte alterations consistent with a stress leukogram were noted for the 750 mg/kg/day group males and females, including lower lymphocyte values (not statistically significant), lower eosinophil values (statistically significant in males) and higher monocyte values (statistically significant in females). Leukocyte parameters were
comparable to the control group at the recovery necropsy.
- Statistically significant changes within the erythron at the primary necropsy included lower hemoglobin and hematocrit values (all treated group males and the 750 mg/kg/day group females); lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) values (all treated group males and the 500 and 750 mg/kg/day group females); higher red cell distribution width (RDW) values (750 mg/kg/day group males
and females); and higher hemoglobin distribution width (HDW) values (750 mg/kg/day group males). There were no changes in the erythron at recovery considered to be test substance-related.
- Test substance-related, statistically significant changes in coagulation parameters were restricted to lower platelet values in the 750 mg/kg/day group females at the primary necropsy. There were no test substance-related changes in coagulation parameters noted at the recovery necropsy.
- There were no other test substance-related effects on hematology or coagulation parameters. However, some statistically significant differences were observed when the control and test substance-treated groups were compared.
- Higher prothrombin time (PT) was noted in the 750 mg/kg/day group females at the primary necropsy; there were no histologic correlates, the magnitude of change was minimal, and values were within the historical control database range.
- Higher erythrocyte count (RBC) values and lower MCV, MCH and reticulocyte values were noted in the 750 mg/kg/day group females at recovery when compared with the control group; there were no histologic correlates and the differences were of minimal magnitude.
- RBC values at recovery were similar to RBC values at the primary necropsy and were within the historical control database range, while control group values were minimally lower than the historical control database range; the finding was attributed to biological variability.
- MCH is a calculated erythrocyte index; lower MHC values were considered a mathematical function of higher RBC values in the recovery group females and not test substance-related.
- Lower reticulocyte values were within the historical control database range; the change was attributed to biological variability.
- Lower MCV values were increased when compared with values at the primary necropsy and were within the historical control database range; lower MCV values correlated with the lower reticulocyte values addressed above, and were attributed to biological variability.
- Statistically significant findings that involved percentage leukocyte differential counts were not itemized above and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
SERUM CHEMISTRY
- Test substance-related, statistically significant higher serum alkaline phosphatase (ALP) values were noted in the 500 and 750 mg/kg/day group males, and higher serum cholesterol (750 mg/kg/day) and triglyceride (500 and 750 mg/kg/day) values were noted in treated group females at the primary necropsy.
- Higher total protein and albumin values were noted in the 750 mg/kg/day group males at the recovery necropsy.
- There were no other test substance-related effects on serum chemistry parameters.
- However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Lower serum chloride values were noted in the 500 and 750 mg/kg/day group males at the primary necropsy; there were no histologic correlates, values were of minimal magnitude change, other electrolyte values were similar to control values, and there was no dose-response relationship.
- Lower serum sorbitol dehydrogenase (SDH) values were noted in the 500 and 750 mg/kg/day group males at the primary necropsy; the change was in a direction of no known toxicologic relevance.
- Lower serum globulin and phosphorus values were noted in the 750 mg/kg/day group females at the primary necropsy; there were no histologic correlates and changes were of minimal magnitude.
- Higher serum glucose was noted in the 750 mg/kg/day group females at the recovery necropsy; there were no histologic correlates and the change was of minimal magnitude.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related, statistically significant changes in urinalysis parameters at the primary necropsy included lower pH values in all treated groups, and higher urine volume values in the 500 (female) and 750 mg/kg/day (male and female) group animals.
- Non-statistically significant higher urine volume values were also noted in the 500 mg/kg/day group males. Urinalysis parameters were comparable to control groups at the recovery necropsy.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
HOME CAGE OBSERVATIONS
- Home cage observations were unaffected by test substance administration.
- A statistically significantly higher number of 750 mg/kg/day group females were noted with feces pellets present and a corresponding statistically significantly lower number of females in this group were noted without feces pellets when compared to the control group. However, the presence of feces is a common finding for rats, and the pellets were formed and showed no indication of diarrhea. In addition, there were no corresponding excreta-related clinical observations noted that would suggest a test substance-related effect. Therefore, these changes were not considered test substance-related.
- There were no other statistically significant differences when the test substance-treated males and females were compared (by sex) to the control group at the study week 12/13 evaluation.
HANDLING OBSERVATIONS
- Handling observations were unaffected by test substance administration.
- There were no statistically significant differences when the test substance-treated males and females were compared (by sex) to the control group at the study week 12/13 evaluation.
OPEN FIELD OBSERVATIONS
- Open field observations were unaffected by test substance administration.
- There were no statistically significant differences when the test substance-treated males and females were compared (by sex) to the control group at the study week 12/13 evaluation.
SENSORY OBSERVATIONS
- Sensory observations were unaffected by test substance administration.
- There were no statistically significant differences when the test substance-treated males and females were compared (by sex) to the control group at the study week 12/13 evaluation.
NEUROMUSCULAR OBSERVATIONS
- Neuromuscular observations were unaffected by test substance administration.
- A statistically significantly lower mean hindlimb footsplay value was noted for the 750 mg/kg/day group males compared to the control group. However, the mean value noted for this group (70.1 mm) was similar to the mean value and within the range of values in the WIL Research historical control data (71.5 mm and 44.2-102.0 mm, respectively) and was not considered test substance-related.
- There were no other statistically significant differences when the test substance-treated males and females were compared (by sex) to the control group at the study week 12/13 evaluation.
PHYSIOLOGICAL OBSERVATIONS
- Test substance-related lower mean body weights were noted at the physiological observations for males in the 750 mg/kg/day group and females in the 500 and 750 mg/kg/day groups compared to the control group. These changes corresponded with body weight effects noted for these groups during the dosing period and were considered test substance-related.
- There were no other test substance-related physiological observations.
- Statistically significantly higher mean body temperatures were noted for the 500 mg/kg/day group males (37.6°C) and the 750 mg/kg/day group females (38.4°C) compared to the control group. However, the mean body temperatures in these groups were similar to the mean values in the WIL Research historical control data (37.4°C and 38.2°C for males and females, respectively), and these changes were not considered test substance-related.
- There were no other statistically significant differences when the test substance-treated males and females were compared (by sex) to the control group at the study week 12/13 evaluation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related higher liver weights were noted in the 500 and 750 mg/kg/day group males and females, and lower thymus weights were noted in the 750 mg/kg/day group males at the primary necropsy.
- Statistically significant higher liver weights (absolute and relative to body and brain weights) were noted in the 500 and 750 mg/kg/day group males and females at the primary necropsy; higher liver weight relative to body weight was also noted in the 250 mg/kg/day group females.
- Statistically significant lower group mean thymus weights (absolute and relative to brain weights) were noted in the 750 mg/kg/day group males at the primary necropsy, which correlated with a stress leukogram and minimal lymphoid depletion noted microscopically; the finding was attributed to a stress response secondary to test substance administration.
- There were no test substance-related changes in organ weights noted at the recovery necropsy.
- There were no other organ weight changes attributed to test item administration.
- Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a test substance-related effect on final body weight, and not directly related to administration of test item (lower epididymis and seminal vesicle/prostate weights in the 750 mg/kg/day group males and higher kidney weights in the 500 and 750 mg/kg/day group males and females at the primary necropsy, and adrenal gland weights in the 750 mg/kg/day group males at the recovery necropsy).
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Review of the gross necropsy observations revealed no findings that were considered to be associated with administration of the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic findings were noted in the liver, thyroid glands, thymus, and ovaries at the primary necropsy (see Table 3, attached).
- Test substance-related hepatocellular hypertrophy was noted in the 500 and 750 mg/kg/day group males and females and the 250 mg/kg/day group females at the primary necropsy. Hepatocytes were diffusely enlarged with no lobular pattern.
- Minimal thyroid gland follicular cell hypertrophy was noted in the 250, 500, and 750 mg/kg/day group males and females at the primary necropsy, characterized by columnar, vacuolated follicular cell cytoplasm and minimally reduced colloid. Both hepatocellular and thyroid follicular cell hypertrophy were considered adaptive changes and not adverse.
- There was no histologic evidence of hepatic and thyroid gland hypertrophy at the recovery necropsy.
- An increased incidence of follicular and/or luteal cysts was noted in the 250, 500, and 750 mg/kg/day group females. The incidence of ovarian cysts was comparable to the control group at the recovery necropsy.
- Thymic lymphoid depletion was noted in the 750 mg/kg/day group males at the primary necropsy, characterized by minimal thinning of the thymic cortex; the finding correlated with a stress leukogram and lower thymus weights, and was consistent with a stress response due to administration of the test substance. Thymic lymphoid depletion was not noted in control or treated group animals at recovery.
- There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
LOCOMOTOR ACTIVITY
- In the 750 mg/kg/day group males and females, test substance-related higher mean overall cumulative total (40.8% and 17.3%, respectively) and ambulatory (70.8% and 24.6%, respectively) counts were noted compared to the control group.
- In addition, the mean values for the 31-40 and 41-50 minute epochs for the 750 mg/kg/day group males were statistically significantly higher than the control group.
- There were no other statistically significant changes for the test substance-treated males and females when compared (by sex) to the control group at the study week 12/13 evaluation.
- Additional values obtained from the 6 epochs evaluated (0-10 minutes, 11-20 minutes, 21-30 minutes, 31-40 minutes, 41-50 minutes, and 51-60 minutes) and the overall 60-minute test session were comparable to the concurrent control values.
- Differences from the control group were slight, not statistically significant when analysed using repeated measures analysis, within the WIL Research historical control data ranges, and/or did not occur in a dose-related manner.
- No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated during study week 12/13.
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
other: motor activity
Remarks on result:
other: all effects considered to be nonadverse at highest dose level of 750 mg/kg/day
Key result
Critical effects observed:
no

ANALYSES OF DOSING FORMULATIONS

- All formulation samples mean recoveries were between 71.9% and 99.7%. With the following exceptions, all recoveries were within the WIL Research SOP range for suspensions (85% to 115%).

- The 50 mg/mL homogeneity and resuspension homogeneity samples contained 78.5% and 75.5% of the target concentration, respectively, and the 26-Aug-2014 concentration sample for this group contained 71.9% of the target concentration.

- The results of the analyses appeared to be time dependent and were consistent with results obtained during the method validation 70-day storage assessment (i.e., formulations at lower concentrations have lower mean percent recovery following longer storage duration.

- For the samples in this study, the number of days from preparation/sampling of the formulations to time of analyses was 7 -37 days.

- Because all formulations were used for dosing within 8 days of preparation, it is believed that mean percent recoveries of the study samples would be increased if they had been analyzed closer to the time of sampling. Thus, animals on this study are believed to have received the appropriate dose concentration/dosage levels during this study.

- The analyzed dosing formulation samples were homogeneous and were stable.

- Results of the analyses of dosing formulations are summarized Tables 1 and 2 (attached).

Conclusions:
Oral gavage administration of test item to Crl:CD(SD) rats for a minimum of 91 consecutive days resulted in effects considered to be nonadverse. Specifically, clinical findings (≥ 250 mg/kg/day), statistically significantly lower body weights (males at 750 mg/kg/day and females at ≥ 500 mg/kg/day), statistically significantly increased motor activity (750 mg/kg/day), alterations (generally statistically significant) in hematology (750 mg/kg/day), serum chemistry (≥500 mg/kg/day), urinalysis (≥250 mg/kg/day), and organ weights (≥ 500 mg/kg/day), macroscopic findings (females at ≥ 500 mg/kg/day), and microscopic findings ≥ 250 mg/kg/day) were considered to be nonadverse. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 750 mg/kg/day.
Executive summary:

GUIDELINE

The objective of the study was to evaluate the potential toxic effects of the test substance when administered via gavage to rats for a minimum of 90 consecutive days in accordance with the OECD Guideline for Testing of Chemicals: Guideline 408. This included evaluation of potential neurotoxicity by functional observational battery and motor activity assessment.

 

METHODS

The test substance in the vehicle (peanut oil) was administered orally by gavage once daily for a minimum of 91 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 250, 500, and 750 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg of body weight for all groups. Groups 1 and 4 each consisted of 15 animals/sex, and Groups 2-3 each consisted of 10 animals/sex. Following a minimum of 91 days of dose administration, 10 rats/sex/group were euthanized; the remaining 5 rats/sex in the control and high-dose groups were euthanized following a minimum 28-day nondosing (recovery) period.

 

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body and food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and locomotor activity data were recorded for all animals during study week 12/13. Ophthalmic examinations were performed during study weeks -2 (during acclimation prior to randomization), 12, and 16. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals at the primary (study week 13) and recovery (study week 17) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

 

RESULTS

All animals survived to the scheduled necropsies. There were no test substance-related effects on food consumption. In addition, there were no test substance-related ophthalmic examination findings.

 

Test substance-related clinical observations were noted during the dosing period and included clear and yellow material around the mouth and yellow material on various body surfaces for the 250, 500, and 750 mg/kg/day group males and females and red material around the mouth for the 250, 500, and 750 mg/kg/day group males and the 500 and 750 mg/kg/day group females. Recovery from these clinical observations was evident during the recovery period.

 

Test substance-related lower body weights were noted for the 750 mg/kg/day group males and the 500 and 750 mg/kg/day group females during the dosing period, resulting in body weights 11.2%, 5.7%, and 5.0% lower than the control group, respectively, at the end of the dosing period. Body weight changes for males and females at 750 mg/kg/day were similar to the control group during the recovery period, but body weights did not fully recover.

 

At the FOB assessment, test substance-related lower body weights were noted for the 750 mg/kg/day group males and the 500 and 750 mg/kg/day group females at the study week 12/13 evaluation. In addition, test substance-related increased total and ambulatory counts were noted for the 750 mg/kg/day group males and females at the study week 12/13 locomotor activity assessment.

 

Test substance-related clinical pathology alterations at the primary necropsy included the following: lower lymphocytes and eosinophils and higher monocytes and red cell distribution width for males and females at 750 mg/kg/day; lower hemoglobin for males at 250, 500, and 750 mg/kg/day and females at 750 mg/kg/day; lower mean corpuscular volume (MCV) for males at 250, 500, and 750 mg/kg/day and females at 500 and 750 mg/kg/day; higher reticulocytes and hemoglobin distribution width (HDW) for males at 750 mg/kg/day; higher alkaline phosphatase for males at 500 and 750 mg/kg/day; higher cholesterol and prothrombin time and lower platelets for females at 750 mg/kg/day; higher triglycerides for females at 500 and 750 mg/kg/day; lower pH for males and females at 250, 500, and 750 mg/kg/day; and higher urine volume and lower specific gravity for males and females at 500 and 750 mg/kg/day. At the recovery necropsy, clinical pathology alterations consisted of higher red blood cells and lower MCV and reticulocytes for females at 750 mg/kg/day and higher total protein and albumin for males at 750 mg/kg/day.

 

Test substance-related macroscopic finding consisted of ovarian cysts for the 500 and 750 mg/kg/day group females at the primary necropsy.

 

Test substance-related higher liver weights were noted for the 500 and 750 mg/kg/day group males and females, and lower epididymides, seminal vesicle/prostate, and pituitary gland weights were noted for the 750 mg/kg/day group males at the primary necropsy. Test substance-related microscopic findings were noted at the primary necropsy and consisted of hepatocellular hypertrophy in the liver for the 500 and 750 mg/kg/day group males and females and the 250 mg/kg/day group females; follicular cell hypertrophy in the thyroid gland for the 250, 500, and 750 mg/kg/day group males and females; and follicular and/or luteal cysts in the ovary for the 250, 500, and 750 mg/kg/day group females.

 

CONCLUSIONS

Oral gavage administration of test item to Crl:CD(SD) rats for a minimum of 91 consecutive days resulted in effects considered to be nonadverse. Specifically, clinical findings (≥ 250 mg/kg/day), statistically significantly lower body weights (males at 750 mg/kg/day and females at ≥ 500 mg/kg/day), statistically significantly increased motor activity (750 mg/kg/day), alterations (generally statistically significant) in hematology (750 mg/kg/day), serum chemistry (≥ 500 mg/kg/day), urinalysis (≥ 250 mg/kg/day), and organ weights (≥ 500 mg/kg/day), macroscopic findings (females at ≥ 500 mg/kg/day), and microscopic findings ≥ 250 mg/kg/day) were considered to be nonadverse. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 750 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Short-term repeated dose oral toxicity

GUIDELINE

The objective of this study was to evaluate the potential toxic effects of the test substance when administered via gavage to rats for 28 consecutive days according to the OECD Guideline for Testing of Chemicals (Guideline 407). This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment.

 

METHODS

The test substance in the vehicle (peanut oil) was administered orally by gavage once daily for 28 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 250, 500, and 1000 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Each group consisted of 5 animals/sex. Following 28 days of dose administration, all surviving rats were euthanized. All animals were observed twice daily for mortality and their moribund state. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body and food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and locomotor activity data were recorded for all surviving animals during study week 3. Clinical pathology parameters (hematology, coagulation, serum chemistry, thyroid hormones, and urinalysis) were analyzed for all surviving rats at the scheduled necropsy (study day 28). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from the animal that died and all animals in the control, 250 (gross lesions only), 500 (gross lesions only), and 1000 mg/kg/day groups at the scheduled necropsy. In addition, the liver, thyroid gland, nasal cavity, testes, and female reproductive tissues (ovaries, uterus, cervix, and vagina) were identified as potential target tissues and were examined from all animals.

 

RESULTS

One 1000 mg/kg/day group female was found dead on study day 18. As there were no clinical or macroscopic findings for this animal, the cause of death was undetermined and its association with the test substance was uncertain. All other animals survived to the scheduled necropsy. There were no test substance-related effects on body weight, alterations in serum chemistry parameters, or macroscopic findings.

Test substance-related clinical observations were noted in the 500 and 1000 mg/kg/day group males and females, generally in a dose-related manner, including clear and/or yellow material around the mouth, yellow material on various body surfaces, and red material around the nose and/or mouth for females. In addition, clear discharge from the eyes was noted for females in the 1000 mg/kg/day group. These clinical findings were noted primarily at the time of dosing and/or 1-2 hours post-dosing as early as study day 3 and generally persisted throughout the study.

Test substance-related clinical observations in the 250 mg/kg/day group included clear material around the mouth in males and females at the time of dosing and/or 1-2 hours post-dosing between study days 12 and 26.

Test substance-related increased incidences of slightly soiled fur were noted for males and females in the 500 and 1000 mg/kg/day groups at the study week 3 functional observational battery evaluation. In addition, test item-related, slightly increased ambulatory activity was noted in males at 500 and 1000 mg/kg/day at the study week 3 locomotor activity assessment.

Test substance-related slightly higher food consumption values were noted in the 500 and 1000 mg/kg/day group males from study days 21 to 27, the 500 mg/kg/day group females from study days 21 to 27, and the 1000 mg/kg/day group females from study days 7 to 27. The higher food consumption values in these groups were considered test substance-related but not adverse due to the absence of correlative body weight gains.

Notable clinical pathology alterations for animals administered 1000 mg/kg/day included higher reticulocytes (males and females), lower platelets (females), lower urine specific gravity (males and females), and higher urine total volume (males and females). Lower urine specific gravity and higher urine total volume was also noted in males at 500 mg/kg/day. There were no correlating microscopic findings.

Lower triiodothyronine (T3) values were noted in the 500 and 1000 mg/kg/day group males; however, there were no corresponding alterations in thyroid stimulating hormone (TSH) and therefore the toxicologic relevance and association of this change with the test substance was uncertain.

Minimal to mild hepatocellular hypertrophy was observed in most of the 1000 mg/kg/day group animals and 2/5 of the 500 mg/kg/day group males, corresponding to higher liver weights noted for 1000 mg/kg/day group animals.. There were no accompanying alterations in clinical pathology or test substance-related microscopic findings indicative of hepatotoxicity; therefore, the change was considered to be nonadverse.

Minimal to mild follicular cell hypertrophy and decreased colloid were observed in the thyroid glands of several of the 500 and 1000 mg/kg/day group males and females and a single 250 mg/kg/day group male. The thyroid changes in these animals were considered to be secondary to enzyme induction rather than evidence of direct thyroid toxicity. Minimal germ cell degeneration and/or tubular degeneration were observed in the testes of the 500 and 1000 mg/kg/day group males as well as a single 250 mg/kg/day group male. A higher incidence of ovarian cysts was observed in the 1000 mg/kg/day group females when compared with the control group, corresponding to the higher ovaries/oviducts weights for these females. The cause of these effects is uncertain. They may reflect a direct toxicity of the test substance on the reproductive tract or may have been secondary to the changes observed in the thyroid gland.

Necrosis of the nasal turbinates was observed in the 1000 mg/kg/day group males and females and the 500 mg/kg/day group males, particularly within the posterior nasal cavity sections (nasal levels III and IV). Minimal necrosis was also observed in the nasal cavity of a single 250 mg/kg/day group male. This nonspecific extensive necrosis and inflammation of the posterior nasal cavity was consistent with gavage-related reflux of the test substance.

Lower thymus weights were also noted in the 250 and 500 mg/kg/day females and 1000 mg/kg/day group males and females. There was no corresponding lymphoid depletion microscopically and most individual animal weights were within the range of the WIL Research historical control data; therefore, the association with test substance administration was uncertain.

 

CONCLUSIONS

Oral gavage administration of test item to Crl:CD(SD) rats for 28 consecutive days resulted in clinical signs (≥250 mg/kg/day), slightly higher food intake (≥500 mg/kg/day), slightly increased motor activity (males at ≥500 mg/kg/day) hematology changes (1000 mg/kg/day), urinalysis alterations (≥500 mg/kg/day), higher liver weights and correlating hepatocellular hypertrophy (1000 mg/kg/day) all of which were considered adaptive and nonadverse, necrosis of the nasal turbinates (most of the 1000 mg/kg/day group animals, some of the 500 mg/kg/day group males, and one 250 mg/kg/day group male) that was considered to be biologically irrelevant to this study, microscopic changes in reproductive organs (testicular germ cell degeneration and/or tubular degeneration and ovarian cysts) and altered estrous cycles (1000 mg/kg/day). The cause of the death of a 1000 mg/kg/day group female could not be determined and its association with the test substance was uncertain. The testicular tubular degeneration observed at 1000 mg/kg/day was considered to be potentially adverse. Based on these results, the no-observed-effect level (NOEL) was less than 250 mg/kg/day and the no-observed-adverse-effect level (NOAEL) was conservatively considered to be 500 mg/kg/day (based upon the uncertainty of the cause of death of one female and testicular tubular degeneration observed at 1000 mg/kg/day).

Subchronic repeated dose oral toxicity

GUIDELINE

The objective of the study was to evaluate the potential toxic effects of the test substance when administered via gavage to rats for a minimum of 90 consecutive days in accordance with the OECD Guideline for Testing of Chemicals: Guideline 408. This included evaluation of potential neurotoxicity by functional observational battery and motor activity assessment.

 

METHODS

The test substance in the vehicle (peanut oil) was administered orally by gavage once daily for a minimum of 91 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 250, 500, and 750 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg of body weight for all groups.

Groups 1 and 4 each consisted of 15 animals/sex, and Groups 2-3 each consisted of 10 animals/sex. Following a minimum of 91 days of dose administration, 10 rats/sex/group were euthanized; the remaining 5 rats/sex in the control and high-dose groups were euthanized following a minimum 28-day nondosing (recovery) period.

 

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body and food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and locomotor activity data were recorded for all animals during study week 12/13. Ophthalmic examinations were performed during study weeks -2 (during acclimation prior to randomization), 12, and 16. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals at the primary (study week 13) and recovery (study week 17) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

 

RESULTS

All animals survived to the scheduled necropsies. There were no test substance-related effects on food consumption. In addition, there were no test substance-related ophthalmic examination findings.

 

Test substance-related clinical observations were noted during the dosing period and included clear and yellow material around the mouth and yellow material on various body surfaces for the 250, 500, and 750 mg/kg/day group males and females and red material around the mouth for the 250, 500, and 750 mg/kg/day group males and the 500 and 750 mg/kg/day group females. Recovery from these clinical observations was evident during the recovery period.

 

Test substance-related lower body weights were noted for the 750 mg/kg/day group males and the 500 and 750 mg/kg/day group females during the dosing period, resulting in body weights 11.2%, 5.7%, and 5.0% lower than the control group, respectively, at the end of the dosing period. Body weight changes for males and females at 750 mg/kg/day were similar to the control group during the recovery period, but body weights did not fully recover.

 

At the FOB assessment, test substance-related lower body weights were noted for the 750 mg/kg/day group males and the 500 and 750 mg/kg/day group females at the study week 12/13 evaluation. In addition, test substance-related increased total and ambulatory counts were noted for the 750 mg/kg/day group males and females at the study week 12/13 locomotor activity assessment.

 

Test substance-related clinical pathology alterations at the primary necropsy included the following: lower lymphocytes and eosinophils and higher monocytes and red cell distribution width for males and females at 750 mg/kg/day; lower hemoglobin for males at 250, 500, and 750 mg/kg/day and females at 750 mg/kg/day; lower mean corpuscular volume (MCV) for males at 250, 500, and 750 mg/kg/day and females at 500 and 750 mg/kg/day; higher reticulocytes and hemoglobin distribution width (HDW) for males at 750 mg/kg/day; higher alkaline phosphatase for males at 500 and 750 mg/kg/day; higher cholesterol and prothrombin time and lower platelets for females at 750 mg/kg/day; higher triglycerides for females at 500 and 750 mg/kg/day; lower pH for males and females at 250, 500, and 750 mg/kg/day; and higher urine volume and lower specific gravity for males and females at 500 and 750 mg/kg/day. At the recovery necropsy, clinical pathology alterations consisted of higher red blood cells and lower MCV and reticulocytes for females at 750 mg/kg/day and higher total protein and albumin for males at 750 mg/kg/day.

 

Test substance-related macroscopic finding consisted of ovarian cysts for the 500 and 750 mg/kg/day group females at the primary necropsy.

 

Test substance-related higher liver weights were noted for the 500 and 750 mg/kg/day group males and females, and lower epididymides, seminal vesicle/prostate, and pituitary gland weights were noted for the 750 mg/kg/day group males at the primary necropsy. Test substance-related microscopic findings were noted at the primary necropsy and consisted of hepatocellular hypertrophy in the liver for the 500 and 750 mg/kg/day group males and females and the 250 mg/kg/day group females; follicular cell hypertrophy in the thyroid gland for the 250, 500, and 750 mg/kg/day group males and females; and follicular and/or luteal cysts in the ovary for the 250, 500, and 750 mg/kg/day group females.

 

CONCLUSIONS

Oral gavage administration of test item to Crl:CD(SD) rats for a minimum of 91 consecutive days resulted in effects considered to be nonadverse. Specifically, clinical findings (≥250 mg/kg/day), statistically significantly lower body weights (males at 750 mg/kg/day and females at ≥500 mg/kg/day), statistically significantly increased motor activity (750 mg/kg/day), alterations (generally statistically significant) in hematology (750 mg/kg/day), serum chemistry (≥500 mg/kg/day), urinalysis (≥250 mg/kg/day), and organ weights (≥500 mg/kg/day), macroscopic findings (females at ≥500 mg/kg/day), and microscopic findings ≥250 mg/kg/day) were considered to be nonadverse. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 750 mg/kg/day.

Justification for classification or non-classification

Administration of test item to rats by gavage in a 90-day repeated dose study via the oral route resulted in no adverse effects at the highest dose of 750 mg/kg bw/ day. Classification for Specific Target Organ Toxicity (repeated exposure) under the terms of Regulation (EC) No 1272/2008 is therefore not required.