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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4th November 2003 to 24th February 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Principles of method if other than guideline:
DEVIATIONS FROM THE PROTOCOL:
This study was conducted in accordance with the protocol and protocol amendments, except for the following.
- On January 5, 2004, tissues for 300 mg/kg/day group male no. 39375 were trimmed according to the protocol-specified tissue list. After the liver was trimmed, it was placed into 70% ethyl alcohol instead of 10% neutral-buffered formalin, and stored overnight. Because ethyl alcohol is frequently used as a tissue preservative, the liver should have been sufficiently preserved for microscopic examination.
These deviations did not negatively impact the quality or integrity of the data nor the outcome of the study.
51
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Phenol, (tetrapropenyl) derivatives (CAS No. 74499-35-7)

Test material purity 100%.

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Forty-six male and 47 female Crl:CD(SD)IGS BR rats in good health were received on November 4, 2003, from Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age at study initiation: approximately 42 days old at receipt. The selected animals were approximately 8.5 weeks old at the initiation of dose administration (November 21, 2003)
- Weight at study initiation: body weight values ranged from 228 g to 288 g for males and from 169 g to 227 g for females.
Individual body weights at randomization were within ± 20% of the mean for each sex.
- Housing: Upon arrival, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).
- Diet (e.g. ad libitum): The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research Laboratories, Inc. The basal diet was provided ad libitum throughout the study, exceptduring the period of fasting prior to blood collection when food, but not water, was withheld.
- Water (e.g. ad libitum): Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering systemwas provided ad libitum throughout the study.
- Acclimation period: All animals were housed for a 17-day acclimation period. During this period, each animal was observed twice daily for mortality and general changes in appearance or behavior.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1°C to 22.3°C
- Humidity (%): 34.2% to 48.4%
- Air changes (per hr): Air handling units were set to provide approximately 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article formulations were weight/volume (test article/vehicle) mixtures. For the test article-treated groups, the appropriate amount of the test article for each group was weighed into a tared, labeled storage container. A predetermined volume of the vehicle was added to each container and the contents were mixed until uniform using a magnetic stirrer. A sufficient volume of the vehicle was added to each container to bring each
formulation to the calibration mark. The test article formulations were prepared approximately weekly as single formulations for each dose level, divided into aliquots for daily dispensation and stored at room temperature. The test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.


VEHICLE
The vehicle used in preparation of the test article formulations and for administration to the control group was 100% pure Mazola® corn oil (exp. dates: January 14, February 4 and March 18, 2005), purchased locally.

For the control group (Group 1), a sufficient volume of corn oil was dispensed into a storage container. The vehicle was dispensed weekly and stirred continuously throughout sampling and dispensation. The vehicle was divided into aliquots for daily dispensation and stored at room temperature. The vehicle was stirred continuously throughout dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of dose administration, test article formulations of a sufficient volume for dose administration to a group of animals for up to 8 days were prepared for the 5 and 300 mg/kg/day groups (1 and 60 mg/mL, respectively). Samples (10 mL) for homogeneity determination were collected from the top, middle and bottom strata of these formulations. In addition, samples similar in size to the amount needed for 1 day of dosing (25 and 40 mL) for stability determinations were collected from the middle stratum of the 1 and 60 mg/mL formulations, respectively, and stored at room temperature for 8 days. Duplicate samples (10 mL each) for concentration analyses were collected during study weeks 0 and 2 from each dosing formulation (including the control group). All samples were stored at room temperature. One set of samples was stored at WIL Research Laboratories, Inc. The second set of samples was shipped under ambient conditions via overnight courier to ChevronTexaco Energy Research and Technology Company, Richmond, California for analyses. The methodology and results of these analyses were presented. The test article formulations were found to be homogeneous, contained the amounts of test article specified in the protocol and were stable for at least 8 days.
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 20, 60, 180, 300 mg/kg/day
Basis:

No. of animals per sex per dose:
Each dose group contained five males and five females except the vehicle group (control) and the highest dosing group both of which contained 10 males and 10 females each.
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: From the vehicle and highest dosing group, 5 animals per sex were sacrificed at the end of the exposure period, with the remaining animals (5 per sex per group) were assigned to a 14 day recovery period.
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed twice daily, at the time of dose administration and approximately 1 to 2 hours following dose administration (designated as 1 hour post-dosing for report presentation purposes). During the recovery period, the animals were observed once daily. All significant findings were recorded. Detailed physical examinations were conducted on all animals weekly, beginning at least 1 week prior to test article administration, and prior to the scheduled necropsies.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded at least weekly, beginning 2 weeks prior to test article administration (study week -2). Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded prior to the scheduled necropsies.

FOOD CONSUMPTION:
Individual food consumption was recorded weekly, beginning 1 week prior to test article administration (study week -1). Food intake was calculated as g/animal/day for the corresponding body weight intervals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from all animals at the scheduled necropsies (study weeks 4 and 6). The animals were fasted overnight prior to the collection of blood samples.
Blood was collected from the vena cava at the time of necropsy.
- Animals fasted: Yes
- How many animals: All
- Parameters examined:
HEMATOLOGY
Total Leukocyte Count (White Cells)
Erythrocyte Count (Red Cells)
Hemoglobin
Hematocrit
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Hemoglobin Concentration (MCHC)
Platelet Count (Platelet)
Prothrombin Time (Pro Time)
Activated Partial Thromboplastin
Time (APTT)
Reticulocyte Count
Percent (Reticulocyte)
Absolute (Retic Absolute)
Differential Leukocyte Count -
Percent and Absolute
-Neutrophil
-Lymphocyte
-Monocyte
-Eosinophil
-Basophil
Platelet Estimate
Red Cell Morphology (RBC Morphology)

SERUM CHEMISTRY
Albumin
Total Protein
Globulin [by calculation]
Albumin/Globulin Ratio (A/G Ratio) [by calculation]
Total Bilirubin (Total Bili)
Urea Nitrogen
Creatinine
Alkaline Phosphatase (Alkaline Phos’tse)
Alanine Aminotransferase (Alanine Transfer)
Aspartate Aminotransferase (Aspartat Transfer)
Gamma Glutamyltransferase (Glutamyl Transfer)
Glucose
Total Cholesterol (Cholesterol)
Calcium
Chloride
Phosphorus
Potassium
Sodium
Triglycerides (Triglyceride)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected from all animals at the scheduled necropsies (study weeks 4 and 6).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional observational battery (FOB) observations were recorded for five animals/sex/group prior to the initiation of dose administration, and during study weeks 3 and 5 (recovery period). Testing was performed by the same technicians, whenever possible, without knowledge of the animal group assignment. The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB with one exception; home cage observations were performed in the animal room.
- Battery of functions tested:
HOME CAGE OBSERVATIONS
Posture
Convulsions/Tremors
Feces consistency
Biting
Palpebral (eyelid) closure

HANDLING OBSERVATIONS
Ease of removal from cage
Lacrimation/Chromodacryorrhea
Piloerection
Palpebral closure
Eye prominence
Red/Crusty deposits
Ease of handling animal in hand
Salivation
Fur appearance
Respiratory rate/character
Mucous membranes/Eye/Skin color
Muscle tone

OPEN FIELD OBSERVATIONS (EVALUATED OVER A 2-MINUTE OBSERVATION PERIOD)
Mobility
Rearing
Convulsions/Tremors
Grooming
Bizarre/Stereotypic behavior
Time to first step (seconds)
Gait
Arousal
Urination/Defecation
Gait score
Backing

SENSORY OBSERVATIONS
Approach response
Startle response
Pupil response
Forelimb extension
Air righting reflex
Touch response
Tail pinch response
Eyeblink response
Hindlimb extension
Olfactory orientation

NEUROMUSCULAR OBSERVATIONS
Hindlimb extensor strength
Hindlimb foot splay
Grip strength-hind and forelimb
Rotarod performance

PHYSIOLOGICAL OBSERVATIONS
Catalepsy
Body temperature
Body weight

OTHER:
LOCOMOTOR ACTIVITY
Observations were recorded for five animals/sex/group prior to the initiation of dose administration, and during study weeks 3 and 5 (recovery period). Locomotor activity, recorded after the completion of the FOB, was measured automatically using the San Diego Instruments, Inc., Photobeam Activity System (San Diego Instruments, Inc., San Diego, California). This personal computer-controlled system utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity. The testing of treatment groups was done according to replicate sequence. Each animal was tested separately. Data were collected in 5-minute epochs and the test session duration was 60 minutes. These data were compiled as four 15-minute sub-sessions for tabulation.
Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of one photobeam) and ambulatory motor activity (interruption of two or more consecutive photobeams).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. A complete necropsy was conducted on all animals. Animals were euthanized by isoflurane anesthesia 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.

HISTOPATHOLOGY: Yes. After fixation, protocol-specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides and stained with hematoxylin and eosin. A second slide of the testes and epididymides was also prepared using a Periodic Acid-Schiff (PAS) stain.
Microscopic examination was performed on appropriate tissues from all animals in the control and 300 mg/kg/day groups at the scheduled primary necropsy. Gross lesions were examined from all animals in all groups. If test article-related microscopic findings were observed at 300 mg/kg/day at the primary necropsy, the same tissues were examined from the 180 mg/kg/day group and so forth until no test article-related effects were observed, as well as from the recovery animals.
At the primary necropsy, the prostate, seminal vesicles, testes and coagulating glands were examined from all males in the 60 and 180 mg/kg/day groups, the epididymides were examined from all males in the 180 mg/kg/day group and the ovaries were examined from all females in the 60 and 180 mg/kg/day groups. The adrenal glands were examined from all males in the 5, 20, 60 and 180 mg/kg/day groups and all females in the 180 mg/kg/day group. The liver was examined from the 20, 60 and 180 mg/kg/day group males and the 60 and 180 mg/kg/day group females. The thyroid glands were examined from the 5, 20, 60, and 180 mg/kg/day group males and the 60 and 180 mg/kg/day group females. At the recovery necropsy, the aforementioned tissues were examined from all animals, with the exception of the pituitary gland, which was examined in males only.
Statistics:
All statistical tests were performed using appropriate computing devices or programs.
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was two or less. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ±1 in the last significant figure.
Body weight, body weight change, food consumption, continuous functional observational battery (FOB), locomotor activity, clinical pathology and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test (Steel and Torrie, 1980). Clinical pathology values for white blood cell types that occur at a low incidence (i.e., monocytes, eosinophils and basophils) were not subjected to statistical analysis.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived to the scheduled necropsies. Test article-related evidence of excessive salivation (clear material around the mouth and/or ventral neck) was noted between study days 9 and 26 in the 180 and 300 mg/kg/day group males and females and did not persist during the recovery period. In addition, urogenital staining (yellow material around the urogenital area) was noted occasionally 1 hour post-dosing during study days 11 through 26 in the 60, 180 and 300 mg/kg/day group females. There were no other test article-related clinical observations. All other observations were noted with similar incidence in the control group, were limited to single animals, were not observed in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN
Test article-related lower mean body weight gains were noted throughout the dosing period in the 180 and 300 mg/kg/day group males. Mean body weight gains in the 180 and 300 mg/kg/day group males were statistically significantly (p<0.05 or p<0.01) lower than the control group values during study weeks 1 to 4. As a result, mean body weights for males in these groups were significantly (p<0.05) lower (9% to 13%) than the control group values at study weeks 3 and/or 4. Mean cumulative body weight gains in the 180 and 300 mg/kg/day group males were significantly (p<0.01) lower than the control group values during study intervals 0 to 3 and 0 to 4. During study weeks 4 to 5 and 5 to 6, mean body weight gain in the 300 mg/kg/day group males showed evidence of recovery. Although mean cumulative body weight gain in the 300 mg/kg/day group remained significantly (p<0.05) lower than the control group values during study interval 0 to 5, a significantly (p<0.05) higher mean body weight gain was noted during study week 5 to 6 in the 300 mg/kg/day group males. By study week 6, the mean body weight for the 300 mg/kg/day group males was only 4% lower than the control group value. There were no other test article-related effects on body weights.
In females, a significantly (p<0.05) lower mean body weight gain was also noted during study week 1 to 2 in the 300 mg/kg/day group. However, mean body weights and cumulative body weight gains in the 300 mg/kg/day group females were comparable to the control group throughout the study.

FOOD CONSUMPTION
Test article-related lower mean food consumption was noted in the 180 and 300 mg/kg/day groups during the dosing period. Slightly lower mean food consumption was noted throughout the dosing period (study weeks 0 to 4) in the 180 and 300 mg/kg/day group males and during study week 0 to 1 in the 180 and 300 mg/kg/day group females; differences from the control group were statistically significant (p<0.05) for the 180 mg/kg/day group males during study week 2 to 3 and for the 300 mg/kg/day group females during study week 0 to 1. Although not statistically significant, compensatory increases in food consumption were noted in the 300 mg/kg/day group males during the recovery period (study weeks 4 to 5 and 5 to 6). There were no other test article-related effects on food consumption.

HAEMATOLOGY
Potentially test article-related effects on hematology parameters were noted in the 180 and 300 mg/kg/day group females at the primary necropsy. Several statistically significant (p<0.05 or p<0.01) differences were noted when the control and test article-treated groups were compared. At the primary necropsy, mean hemoglobin and hematocrit levels in the 180 and 300 mg/kg/day group females and mean percent lymphocyte count in the 300 mg/kg/day group females were lower than the control group values. Although not statistically significant, mean absolute and percent reticulocyte counts in the 180 and 300 mg/kg/day group females were higher than the control group values at the primary necropsy.

CLINICAL CHEMISTRY
There were no test article-related changes in serum chemistry parameters. However, several statistically significant (p<0.05 or p<0.01) differences were noted when the control and test article-treated groups were compared. At the primary necropsy, mean aspartate aminotransferase (AST) level in the 180 mg/kg/day group males and mean cholesterol level in the 180 and 300 mg/kg/day group females were lower than the control group values. In addition, mean triglyceride level in the 180 and 300 mg/kg/day group males was higher than the control group values. Gamma glutamyltransferase (GGT) values were higher in the 300 mg/kg/day males and slightly elevated in the 180 mg/kg/day males and females and 300 mg/kg/day females at study week 4. However, in the absence of concurrent increases in alkaline phosphatase (ALP), the significance of the higher GGT values is uncertain. At the recovery necropsy, mean cholesterol level in the 300 mg/kg/day group males was higher and mean chloride level in the 300 mg/kg/day group females was lower than the control group values. Due to the lack of consistent trends between the sexes and between the primary and recovery necropsies, these changes were considered incidental and unrelated to test article administration.

NEUROBEHAVIOUR
Home cage observations, open field observations, sensory observations, neuromuscular observations and physiological observations were unaffected by test article administration. There were no statistically significant changes for the test article-treated males and females when compared to the control group at the study week 3 and 5 evaluations.

ORGAN WEIGHTS
Test article- and dose-related effects on seminal vesicle, prostate, testis, epididymis, adrenal gland, ovary and liver weights were noted in the 180 and 300 mg/kg/day groups. A potentially test article-related effect on heart weights was noted in the 180 and 300 mg/kg/day group males. At the study week 4 primary necropsy, mean absolute and relative (to final body weight and to brain weight) seminal vesicle, prostate, testis (300 mg/kg/day group only) and epididymis weights in the 180 and/or 300 mg/kg/day group males were generally significantly (p<0.05 or p<0.01) lower than the control group values. In addition, mean absolute and relative (to final body weight and to brain weight) adrenal gland weights were higher than the control group values in the 180 and 300 mg/kg/day group males and/or females; differences from the control group were statistically significant (p<0.05 or p<0.01) for the 180 and 300 mg/kg/day group males and the 300 mg/kg/day group females (relative to final body weight).

GROSS PATHOLOGY
Test article-related macroscopic findings of small reproductive organs (testes, prostate, seminal vesicles, epididymides and/or coagulating glands) were noted in the 180 and 300 mg/kg/day group males at the primary necropsy. No similar macroscopic findings were observed at the recovery necropsy. All other macroscopic changes noted were considered to be spontaneous and/or incidental in nature and unrelated to test article administration. There were no macroscopic findings in males in the 5, 20 and 60 mg/kg/day groups or in females at any dose level.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test article-related microscopic findings were noted in the liver, adrenal glands, thyroid glands, seminal vesicles, prostate, testes, epididymides, coagulating gland and/or ovaries in the 5, 20, 60, 180 and 300 mg/kg/day groups at the primary necropsy. In the liver, minimal to mild centrilobular hepatocellular hypertrophy was noted in the 60, 180 and 300 mg/kg/day group males and the 180 and 300 mg/kg/day group females.
In the adrenal glands, cortical hypertrophy was noted in the 20, 60, 180 and 300 mg/kg/day group males and the 300 mg/kg/day group females. The severity and/or incidence of this change tended to increase with dose.
In the thyroid glands, hypertrophy of follicular cells was noted at a low incidence in the 5, 20, 60, 180 and 300 mg/kg/day group males.
Decreased secretion in the male accessory sex glands (seminal vesicles, prostate and coagulating glands) was noted in all males in the 180 and 300 mg/kg/day groups at the primary necropsy. As a component of the decreased fluid within these glands, the lining epithelial cells were reduced in size (atrophy).
In the testes, maturation depletion of germ cells and interstitial cell atrophy were noted in the 180 and 300 mg/kg/day group males. The severity and incidence of these findings tended to increase with dose.
In the ovary, decreased corpora lutea were noted in 2/5 and 3/5 females in the 180 and 300 mg/kg/day groups, respectively.
At the recovery necropsy, test article-related microscopic findings persisted in the seminal vesicles, testes, epididymides and ovaries in the 300 mg/kg/day group.

OTHER FINDINGS
Locomotor activity patterns (mean ambulatory and total motor activity counts) were unaffected by test article administration.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Organ weight and/or microscopic findings in the reproductive organs persisted to the recovery necropsy
Dose descriptor:
NOEL
Effect level:
< 5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Microscopic finding of follicular cell hypertrophy in the thyroid glands in one male from this group at the primary necropsy
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Clinical observations noted at 60 mg/kg/day

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the no-observed-effect level (NOEL) for oral (gavage) administration of tetrapropenyl phenol to male rats for 28 consecutive days was less than 5 mg/kg/day due to a microscopic finding of follicular cell hypertrophy in the thyroid glands in one male from this group at the primary necropsy. For female rats, the NOEL was 20 mg/kg/day due to clinical observations noted at 60 mg/kg/day. Organ weight effects and microscopic findings in the adrenal gland were noted in males of the 20 mg/kg/day group and above. The microscopic findings in the thyroid and adrenal glands of the 300 mg/kg/day group did not persist to the recovery necropsy. Organ weight effects and microscopic findings in the liver were noted at 60 mg/kg/day and above and organ weights and/or microscopic findings in the reproductive organs (ovaries, seminal vesicles, prostate, coagulating glands, epididymides and/or testes) were noted at doses of 180 mg/kg/day and above. Because the organ weight and/or microscopic findings in the reproductive organs persisted to the recovery necropsy, the no-observed-adverse-effect level (NOAEL) was considered to be 60 mg/kg/day for males and females.
Executive summary:

The test material in the vehicle, corn oil, was administered orally by gavage once daily for a minimum of 28 consecutive days to five groups of Crl:CD (SD)IGS BR rats. Dosage levels were 5, 20, 60, 180 and 300 mg/kg/day. A concurrent control group received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Functional observational battery and locomotor activity data were recorded for five animals/sex/group prior to the initiation of dose administration, and during study weeks 3 and 5 (recovery period). Following 28 days of dose administration, five rats/sex/group were euthanized; the remaining animals in the control and 300 mg/kg/day groups were euthanized following a 14-day nondosing (recovery) period.

Clinical pathology evaluations (hematology and serum chemistry) were performed on all rats euthanized at the primary (study week 4) and recovery (study week 6) necropsies.

Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically.

All animals survived to the scheduled necropsies. There were no test article-related effects on functional observational battery parameters or locomotor activity. There were no test article-related effects on clinical pathology parameters.

Evidence of excessive salivation (clear material around the mouth and ventral neck) in the 180 and 300 mg/kg/day group males and females and urogenital staining in the 60, 180 and 300 mg/kg/day groups were limited to the dosing period. Lower body weights,

body weight gains and/or cumulative body weight gains were noted in the 180 and 300 mg/kg/day group males during study weeks 0 to 4; these changes showed evidence of recovery during study weeks 5 and 6. Slightly lower food consumption was also noted in

the 180 and 300 mg/kg/day group males throughout the dosing period and in the 180 and 300 mg/kg/day group females during study week 0 to 1. Potentially test article-related lower hemoglobin and hematocrit levels and/or percent lymphocyte counts and higher

absolute and percent reticulocyte counts were observed in the 180 and 300 mg/kg/day group females at the primary necropsy.

Macroscopically, small testes, prostate, seminal vesicles, epididymides and/or coagulating glands were noted in the 180 and 300 mg/kg/day group males at the primary necropsy. Lower absolute and relative seminal vesicle, prostate, testis, epididymis and

ovary weights were noted in the 180 and 300 mg/kg/day groups. In addition, higher absolute and/or relative liver and adrenal gland weights were noted in the 20, 60, 180 and 300 mg/kg/day groups at the primary necropsy. At the recovery necropsy, lower seminal

vesicle, prostate, testis, epididymis and ovary weights persisted in the 300 mg/kg/day group, suggesting a residual effect or incomplete recovery.

At the primary necropsy, microscopic findings attributed to test article administration were noted in all test article-treated groups. Findings at 180 and 300 mg/kg/day included decreased secretion in the seminal vesicles, prostate and coagulating glands, maturation

depletion of germ cells and interstitial cell atrophy in the testes, luminal cellular debris and/or hypospermia in the epididymides and decreased corpora lutea in the ovaries.

Findings at 60 mg/kg/day and above included centrilobular hepatocellular hypertrophy and hepatocellular vacuolization in the liver. In addition, cortical hypertrophy in the adrenal glands extended to the 20 mg/kg/day males and hypertrophy of follicular cells in

the thyroid gland extended to the 5 mg/kg/day group males. At the recovery necropsy, microscopic changes persisted in the seminal vesicles, testes, epididymides and ovaries in the 300 mg/kg/day group.

Because the organ weight and/or microscopic findings in the reproductive organs persisted to the recovery necropsy, the no-observed-adverse-effect level (NOAEL) was considered to be 60 mg/kg/day for males and females.