2,6-bis(1,1-dimethylethyl)-4-(phenylenemethylene)cyclohexa-2,5-dien-1-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare Guidelines (Notification No. 24, Pharmaceutical Affairs Bureau, September 11, 1989, and as amended by Notification No. 88, August 10, 1993) and with Japanese Substance Law (no. 700), MHW (1039) and MITI (1014).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Substituted Quinone Methide

IUPAC Name:

Substituted Quinone Methide

Details on test material:

- Name of test material (as cited in study report): TKA 45000
- Physical state: Orange to yellow coloured powder
- Lot/batch No.: Production Run #001
- Storage condition of test material: Stored at room temperature

Test animals

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague-Dawley Crl:CD®BR rats was received on July 21, 1998, from Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age at study initiation: Approximately 6 weeks of age.
- Weight at study initiation: 170 to 206 g for males and 122 to 164 for females.
- Fasting period before study: None.
- Housing: Animals of the same sex were housed two per cage in stainless-steel, hanging, wire mesh cages measuring 24.2 x 22.0 x 17.3 cm (d x w x h). Following assignment to study, each animal was individually housed.
- Diet (e.g. ad libitum): Certified rodent diet was available ad libitum during the acclimation and study periods.
- Water (e.g. ad libitum): Tap water, via an automatic watering system, was available as libitum.
No contaminants were known to be present in the diet or water at levels which might interfere with the study.
- Acclimation period: Approximately 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to maintain range of 18 to 26°C.
- Humidity (%): 50 ± 20%
- Air changes (per hr): Ten or greater iar changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle.

IN-LIFE DATES: From: August 3rd 1998 To: September 1st 1998 (Terminal Sacrifice), September 15th 1998 (Recovery Sacrifice).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared at least once weekly for the purpose of dosage calculations. Dose concentrations were based on the test material as supplied. All dose preparations were stored at room temperature until dosed. The formulations were stable for up to 11 days at room temperature.

For each dosing formulation, the appropriate amount of test substance was weighed on an appropriate analytical balance (mg) and transferred to a pre-calibrated beaker. The vehicle, corn oil, was added in small amounts and mixed into a paste. The corn oil was added to the paste until the desired volume was achieved. The formulations were mixed on a magnetic stirrer, sonicated until solution was achieved, subsequently transferred to jars that were suitable for the dosing procedure, and sent to the animal laboratory.

METHOD OF ADMINISTRATION:
Animals were given the appropriate dosing formulation via oral gavage once daily for 29 days at the same time each day (±30 minutes), with the exception of the days on which the expanded clinical observations were performed. On those days, the animals were dosed after the expanded clinical observations were completed. For dosing, the animal was held securely to immobilize the head and administered the appropriate volume of test solution or control material via a laboratory gavage needle. Dosage levels were based on the most recently recorded body weight. Dose volume was based upon the most recently recorded body weight and the dose factor of 10 mL/kg. The formulations were stirred for at least 10 minutes prior to dosing and during the dosing procedure. Treatment continued through the day prior to the scheduled necropsy or through Day 29 for the animals designated to undergo recovery.

VEHICLE
Corn oil was used as the vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity.
Evaluation for homogeneity was performed on the anticipated low- and high-dose formulations (1 and 100 mg/mL, respectively) prior to initiation of dosing. Analyses were performed in duplicate from samples taken from the top, middle, and bottom of the formulations. The remaining set of samples was stored frozen at -20°C and retained pending acceptance of the analytical results.

Stability.
Prior to initiation of dosing, samples from the anticipated low- and high-dose formulations (1 and 100 mg/mL, respectively) were analyzed to assess Day 4, 7, and 11 stability using samples taken from the middle of the homogeneity mix. Analyses were performed in duplicate from samples stored at room temperature.

Routine Concentration.
A 20-mL sample of each formulation was taken from the middle of each batch at Weeks 1, 2, 3, and 4 for analysis. Two replicates were taken from the 20 mL sample and analyzed. The residual aliquot was discarded.


Results:
Homogeneity analyses (low- and high-dose levels) indicated that the test material was homogeneously mixed.

Results of stability analyses indicated that the formulations were stable at 4, 7, and 11 days at room temperature. All values were within 4.3% of initial concentration.

Results of routine concentration analyses indicated that all formulations were within 9% of target.

Duration of treatment / exposure:

29 days.

Frequency of treatment:

Animals were given the appropriate dosing formulation via oral gavage once daily for 29 days at the same time each day.

Doses / concentrations

Remarks:

Doses / Concentrations:
Control (0), 10, 30, 100 and 300 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group 1 - Control: 10 males and 10 females.
Group 2 - Low dose (10 mg/kg/day): 5 males and 5 females.
Group 3 - Mid dose (30 mg/kg/day): 5 males and 5 females.
Group 4 - Mid-high dose (100 mg/kg/day): 5 males and 5 females.
Group 5 - High dose (300 mg/kg/day): 10 males and 10 females.

The last five animals/sex in Group 1 and 5 were designated to undergo a 14-day postreatment recovery period.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses were slected based upon the results of a 7-day gavage range finding study.

- Rationale for animal assignment (if not random):
Animals were initially accepted into the randomization pool based upon physical and ophthalmic examinations; animals with findings were eliminated from the randomization pool. A total of 70 rats (35/sex) was assigned to study using a computerized weight- randomization program, which first eliminated the animals with extreme body weights, then selected the random assignment that produced homogeneity of variance and means. At randomization, the weight variation of the animals selected did not exceed ±2 standard deviations of the mean body weight for each sex, and the mean body weight for each group of each sex was not statistically different. During the randomization process, each study animal was assigned a unique number and individually housed. An implantable microchip identification device (inserted subcutaneously in the lumbar region) was used to permanently identify each animal.

Positive control:

No positve control group.

Examinations

Observations and examinations performed and frequency:

Clinical Observations:
The rats were observed twice daily (a.m. and p.m., at least 6 hours between observations) for evidence of mortality and moribundity. Additional signs of poor health or abnormal behavior were recorded as they were observed.
On each day of dosing, each animal was observed approximately 1 hour post-dose
(+15 minutes). During the recovery period, the daily cage side observations were performed with the morning mortality check. Findings were recorded as they were observed.
Once before treatment and weekly thereafter, each animal was removed from its cage and examined for signs of poor health or abnormal behavior. Findings were recorded for each animal.

Body Weights:
Individual body weights were recorded prior to treatment on the first day of dosing, and weekly thereafter.

Food Consumption:
Individual food consumption was measured and recorded weekly.

Opthalmic Examinations:
Indirect ophthalmic examinations were performed prior to treatment and prior to terminal sacrifice. Findings were to be documented photographically. Mydriacyl® (1%) was used as the mydriatic agent.

Expanded Clinical Observations:
A battery of behavioral tests and observations designed to measure various aspects of sensory and motor functions was conducted on all animals once prior to dosing, once weekly during the 29 days of dosing, and during the second week of recovery (Week 6). Additional detailed clinical observations were performed on all animals during the fourth week of treatment and during the second week of recovery (Week 6).
A description of the criteria for each observation is as follows:

Hand-Held Observations: The hand-held observations were made while the tester restrained the animal gently in on or both hands.
- reactivity to handling, palpebral closure, excessive salivation, appearance of fur, muscle tone, exophthalmos, excessive lacrimation, respiration, piloerection, pupillary status.

Open-Field Observations: Each animal was placed into a open-field arena for approximately 1 minute. The open field was large enough for the rat to walk freely so that the observer could evaluate gait. The open field provided an unobstructed view of the animal.
- locomotor activity, posutre, gait abnormalities, other unusual behaviour.

Elicited Behaviours:
Each animal was tested for auditory reactivity before removal from the open-field arena. Following this test, each animal was removed from the open-field arena and evaluated for all subsequent elicited behaviors while the tester gently restrained the animal.
-auditory reactivity, proprioceptive positioning reaction, nociceptive reflex, pupillary response, pinna response, pupillary status, grip strength.

Grip Strength: Animals were tested using an apparatus consisting of a grasping bar or grid attached to a strain gauge. The animal was allowed to grip the grasping bar or grid and was pulled away from the bar or grid until the grip was broken. Grip strength was recorded for both forelimb and hindlimb grips. Three trials each for the forelimbs and hindlimbs were done. The results of each trial were recorded in grams of force and the mean grip strength was calculated.

Nociceptive Reflex: The animal's tail was place into an automated system. A heat stimulus was applied to the tail. The time (in seconds) for the tail to move away from the nociceptive stimulus was recorded.

Clinical Pathology:
Prior to each clinical sampling, all surviving animals were placed in urine collection racks and fasted overnight (with water available). Blood samples for hematology, coagulation, and serum chemistry evaluations were obtained prior to terminal and recovery sacrifices via jugular venipuncture. Urine specimens were obtained during the overnight fast in individual urine collection cages.
The following parameters were determined:

Hematology:
- red blood cell (erythrocyte) count
- hemoglobin
- hematocrit
- red cell distribution width
- mean corpuscular volume
- mean corpuscular hemoglobin
- mean corpuscular hemoglobin concentration
- platelet count
- white blood cell (leukocyte) count
- differential blood cell count
- blood cell morphology

Coagulation:
- activated partial thromboplastin time
- fibrinogen
- prothrombin time

Serum Chemistry:
glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin, globulin ratio, cholestrol, triglycerides, total bilirubin, alkaline phosphatase, alanine aminotransferase, gamma glutamyltransferase, aspartate aminotransferase, calcium, inorganic phosphorus, sodium, potassium, chloride.

Urinalysis:
appearance, pH, protein, urobilinogen, glucose, ketones, bilirubin, blood, microscopic examination of sediment.

Sacrifice and pathology:

Necropsy:
After 29 days of treatment, the first five surviving animals/sex/group were fasted overnight, weighed, anesthetized with sodium pentobarbital, exsanguinated, and necropsied. After at least 14 days of recovery, the remaining animals/sex in Groups 1 and 5 were fasted overnight, weighed, anesthetized with sodium pentobarbital, exsanguinated, and necropsied. A necropsy was performed on each animal by appropriately trained personnel using procedures approved by board-certified pathologists, and all findings were recorded. The necropsy included examination of the following:
- all orifices
- cranial cavity
- external surface of the brain; the external surface of the spinal cord and cut surfaces of the brain and spinal cord were examined at tissue trimming.
- cervical tissues and organs
- thoracic, abdominal and pelvi cavities and viscera
- external surface of the body
- nasal cavity and paranasal sinuses.

Organ Weights:
At each scheduled sacrifice, the following organs (when present) were weighed after careful dissection and trimming of fat and other contiguous tissue:
- adrenal, ovary, liver, heart, spleen, kidney, testis with epididymis, brain, thymus, thyroid gland with parathyroid.

Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.

Tissue Preservation:
The following tissues from each animal were preserved in 10% neutral-buffered formalin:
Adrenal, [nasopharynx], aorta, ovary, brain (cerebrum, cerebellum, pons), pancreas, cecum, pituitary gland, colon prostate gland, duodenum, rectum, esophagus, sciatic nerve, [eye with optic nerve] [seminal vesicle] , [femur with bone marrow (articular surface ofthe distal end)], [harderian gland], heart, ileum (with Peyer's patches), jejunum, Kidney,[larynx], lesions, liver, lung, mammary gland (females only), mandibular lymph node, [mandibular salivary gland], mesenteric lymph node, [skeletal muscle, thigh], [skin], spleen, spinal cord (cervical, mid-thoracic and lumbar), stomach, sternum with bone marrow, [sublingual salivary gland], testis with epididymis, thymus, thyroid with parathyroid, [tongue], trachea, urinary bladder, uterus with cervix and vagina

Histopathology:
The preserved tissues from the terminal-sacrifice animals in Groups 1 and 5 were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. The slides from tissues in brackets were examined only if indicated by signs of toxicity or target organ involvement.

Macroscopic lesions, lungs, liver, and kidney were also examined microscopically from each terminal-sacrifice animal at the low, mid, and mid-high doses. The liver was identified as a target organ and examined microscopically from recovery-sacrifice animals.

Other examinations:

None.

Statistics:

Body weights, body weight change, weekly food consumption, total food consumption, clinical pathology (except hemolysis and cellular morphology gradings and routine urinalysis data), fasted terminal body weight, and organ weight data of the treated groups were compared statistically to the data from the same sex of the control group.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Deaths not related to test material administration, but gavage-related trauma.

Mortality:

mortality observed, treatment-related

Description (incidence):

Deaths not related to test material administration, but gavage-related trauma.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See details on results for discussion.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See details on results for discussion.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See detaails on results for discussion.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details on results for discussion.

Histopathological findings: neoplastic:

no effects observed

Details on results:

In-life Observations:
Mortality:
One female and one male given 300 mg/kg/day died on Days 6 and 29, respectively, with no clinical observations noted for either animal. Another male, given 300 mg/kg/day was noted as pale on Day 29 and was found dead on Day 30. These deaths were not related to test material administration, as microscopic examination of the tissues indicated that death was due to gavage-related trauma as a result of the difficulty in dosing.

Clinical Observations:
One Group 5 male was noted to have audible respiration on Days 29, 36, and 43; this animal survived to scheduled sacrifice. Group 5 animals were observed to struggle against dosing at the beginning of Week 2, with resultant backwash out of the mouth noted. Additional care and handling reduced the struggling and the backwash.

There were no other remarkable clinical observations noted for animals in the control or test groups throughout the course of the study.

Expanded Clinical Observations:
None of the observations were consistent with a test article-related effect on neurobehavioral functions. There were no marked differences in the quantitative parameters between the control and treated rats or abnormal observations in the subjective evaluations.

Body Weights:
There were no significant differences in mean body weight values throughout the course of the study. The Group 5 female mean body weight change value was significantly increased from the control value at the Day 1-8 interval.

Food consumption:
There were no significant differences in mean food consumption values throughout the course of the study.

Ophthalmic Findings:
An incidental finding in the eye of one animal was photographed by the ophthalmologist. The photograph was damaged due to technical problems.
None of the observations noted at Day 29 were attributed to test material administration; therefore, examinations were not performed on the recovery animals.

Clinical Pathology:

The mean values for prothrombin time (PT) and activated partial tissue thromoplastin time (APTT) were higher in Group 4 males and Group 5 animals, significantly for the Group 5 males, at Day 30; the increases were of low magnitude for the females. The values remained slightly higher in Group 5 animals relative to control values at Day 44, significantly for the males. The mechanism of the increases is not apparent from the data examined (clinical observations, body weights, body weight changes, food consumption, necropsy findings, organ weights, and remaining clinical laboratory data), but the increases are attributed to treatment.

The platelet counts were >10E6/µL in each Group 5 animal, with a significantly increased mean noted for the female platelet count at Day 30. The mechanism behind this increase is not apparent from the data examined, but it is suspected to be an effect from the administration of the test material; the Group 5 values at Day 44 were comparable to those of control animals.

The mean alanine aminotransferase values were slightly higher in the Group 5 animals at Day 30, significantly for the females. Although of low magnitude, these increases may be related to treatment and, for the females, were accompanied by higher liver weights at terminal sacrifice.

The remaining changes in the clinical pathology data were considered incidental to test material administration.

Unscheduled Deaths:
A female given 300 mg/kg/day, died on Day 6. At necropsy, no remarkable observations were recorded. Microscopically, acute inflammation involved the pleura of the thymus and epicardium of the heart. Additionally, foreign material, compatible with foodstuff, was present in the inflammatory exudate around the thymus, and bacteria were evident around the heart. The cause of death was sepsis, which was probably secondary to gavage-related trauma and esophageal perforation.

A male given 300 mg/kg/day, died on Day 29. At necropsy, a large amount of dark-red fluid material was present in the thoracic cavity. Microscopically, there was extensive hemorrhage involving the thymus, and bacteria were evident. Additionally, inflammation involving the esophagus suggested the probability of gavage-related trauma. The cause of death was considered to be sepsis and hemorrhage.

Another male given 300 mg/kg/day, died on Day 30. At necropsy, this animal had a pale liver and a large amount of red fluid in the thoracic cavity. Microscopically, acute inflammation was evident around the heart, lung, and brain. Moderate centrilobular necrosis was present in the liver. This liver lesion was consistent with acute passive congestion associated with cardiac dysfunction. The cause of death was sepsis, which was probably secondary to gavage-related trauma.

Terminal and Recovery Sacrifices:
At necropsy of terminal- and recovery-sacrifice animals, there were no macroscopic observations that appeared to be related to administration of the test material. The few gross findings recorded were randomly scattered among treatment groups, were low in incidence, and are considered to be
common background findings in laboratory rats.

Organ weights:
Increased mean values were noted for the absolute liver weight, liver-to-body weight percentage, and liver-to-brain weight ratio in the females given 300 mg/kg/day (Group 5) and for the liver-to-body weight percentage in the females given 100 mg/kg/day (Group 4). Similar liver weight changes were not observed in treated males. Microscopically, there was an increase in the incidence and severity of periportal vacuolation in the livers of Group 3, 4, and 5 females.

The mean absolute brain weight was increased in the Group 4 females, and the mean absolute heart weight was reduced in the Group 5 males. These organ weight changes are considered incidental to test material administration. No other remarkable organ weight differences were idendtified.

Microscopic Observations:
For females given 30, 100, and 300 mg/kg/day, there was an increase in the incidence of minimal to slight periportal hepatocellular vacuolation in the liver, and this appeared to correlate with the increased mean liver weight in the Group 5 females.
For males, periportal hepatocellular vacuolation did not appear to be treatment-related. In fact, control males had a more-severe periportal vacuolation than did males given the test material. Clinical chemistry results did not indicate any notable hepatic dysfunction in the males or females, and the toxicologic significance of this finding in females did not appear to be adverse. All other histomorphologic findings in the liver are considered incidental to test material administration.

Examination of the livers from the recovery sacrifice animals did not reveal any test material-related changes. The incidence of periportal hepatocellular vacuolation was not remarkably different between Group 5 females and Group 1 females.

No other microscopic diagnoses appeared to be related to administration of the test material. Histopathology findings noted were those frequently seen as background findings in laboratory rats and are considered incidental to test material administration.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based upon the lack of effects after a recovery period, the No-Observable Adverse Effect Level (NOAEL) for the test material in both males and females is 300 mg/kg/day.

2

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Discussion

Three rats that received 300 mg/kg/day (two males, one female) were found dead during the study; due to the clinical observations and the presence of histological evidence, these deaths were determined to be gavage-related. In the surviving animals, there were no clinical observations or behavioral changes which were considered test article-related.

There were no significant differences in body weight or food consumption data indicative of toxicity or test article related ophthalmological findings. Observations at necropsy were those noted routinely in rats of this age.

Test article-related changes noted in the clinical pathology data at the time of the terminal sacrifice included slightly higher mean values for prothrombin time and activated partial thromboplastin time in males that received 100 and 300 mg/kg/day and in females that received 300 mg/kg/day (statistically significant for the males at 300 mg/kg/day).

Alanine aminotransferase was slightly elevated in animals that received 300 mg/kg/day. With the exception of prothrombin time and activated partial thromboplastin time in the males, these values were comparable to the control value after the recovery period. The toxicological relevance of the increases in prothrombin time and activated partial thromboplastin time is unclear, as there was no indication from the remainder of the data that this resulted in adverse events.

Mean liver weights at terminal sacrifice were slightly elevated in the females that received 100 (liver-to-body weight percentage only) and 300 mg/kg/day with increases in the incidence and severity of periportal vacuolation in the livers of the 30, 100, and 300 mg/kg/day females. After the recovery period, the incidence of periportal hepatocellular vacuolation in the 300 mg/kg/day rats was not remarkably different from that of the control group.

In conclusion, when the test material was given to rats once daily by gavage in corn oil for 29 days at a dose level of 10, 30, 100, or 300 mg/kg, there were no adverse effects noted in males given 300 mg/kg/day or less. In females given 30, 100, and 300 mg/kg/day, there was an increase in the incidence and severity of periportal hepatocellular vacuolation and increased liver weights in females given 100 and 300 mg/kg/day; based upon the histopathology, the No-Observable Effect Level (NOEL) is 10 mg/kg/day. Slight increases in alanine aminotransferase were noted in the 300 mg/kg/day animals. After the 14 day recovery period, these differences were not noted between the controls and the rats that received 300 mg/kg/day and therefore, based upon the lack of effects after a recovery period, the No-Observable Adverse Effect Level (NOAEL) for the test material in both males and females is 300 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

When the test material was given to rats once daily by gavage in corn oil for 29 days at a dose level of 10, 30, 100, or 300 mg/kg, there were no adverse effects noted in males given 300 mg/kg/day or less. In females given 30, 100, and 300 mg/kg/day, there was an increase in the incidence and severity of periportal hepatocellular vacuolation and increased liver weights in females given 100 and 300 mg/kg/day; based upon the histopathology, the No-Observable Effect Level (NOEL) is 10 mg/kg/day. Slight increases in alanine aminotransferase were noted in the 300 mg/kg/day animals. After the 14 day recovery period, these differences were not noted between the controls and the rats that received 300 mg/kg/day and therefore, based upon the lack of effects after a recovery period, the No-Observable Adverse Effect Level (NOAEL) for the test material in both males and females is 300 mg/kg/day.

Executive summary:

The purposes of this study was to evaluate the toxicity of a test material when administered daily by gavage to rats for at least 28 days and the reversibility of any toxicity followed by a 14-day post treatment recovery period.

Male and female Sprague-Dawley Crl:CD BR rats were assigned to 5 groups (10/sex/group in Groups 1 and 5, 5/sex/group in Groups 2, 3, and 4). Each group received 10, 30, 100, or 300 mg of test material/kg of body weight (Groups 2 through 5, respectively) by gavage in corn oil once daily for 29 days. Group 1 (control group) received only the corn oil.

Diet and water were provided ad libitum, unless otherwise noted. The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity and once daily for signs of toxicity. Weekly detailed observations (including neurobehavioral examination), body weights, and food consumption measurements were recorded. Ophthalmic examinations were done before initiation of treatment and prior to sacrifice. Blood and urine samples were collected for hematology, clinical chemistry, and urinalysis at scheduled sacrifices. After 29 days of treatment, five rats/sex/group were anesthetized, weighed, exsanguinated, and necropsied; after an additional 14 days of observation, the surviving rats in Groups 1 and 5 were sacrificed. At necropsy, macroscopic observations were recorded, selected organs were weighed, and selected tissues were collected and preserved; animals that died on test were also necropsied. Microscopic examinations were done on tissues from each animal in the control and high-dose groups and from each animal that died or was sacrificed at an unscheduled interval. The liver, lungs, and kidneys were also examined microscopically from each animal in the low-, mid,- and mid-high-dose groups, and the liver was examined from the recovery groups.

Three rats that received 300 mg/kg/day (two males, one female) were found dead during the study; these deaths were determined to be gavage-related. In the surviving animals, there were no clinical observations or behavioral changes which were considered test article-related. There were no significant differences in body weight or food consumption data indicative of toxicity or test article related ophthalmological findings. Observations at necropsy were those noted routinely in rats of this age.

Test article-related, toxicologically relevant, changes noted in the clinical pathology data at the time of the terminal sacrifice included slightly elevated alanine aminotransferase in rats that received 300 mg/kg/day. This value was comparable to the control value after the recovery period.

Mean liver weights at terminal sacrifice were significantly elevated in the females that received 100 (liver-to-body weight percentage only) and 300 mg/kg/day, with noted increases in the incidence and severity of periportal vacuolation in the livers of the 30,

100, and 300 mg/kg/day females. After the recovery period, the incidence of periportal hepatocellular vacuolation in the 300 mg/kg/day rats was not remarkably different from that of the control group.

In conclusion, when the test material was given to rats once daily by gavage in corn oil for 29 days at a dose level of 10, 30, 100, or 300 mg/kg, there were no adverse effects noted in males given 300 mg/kg/day or less. In females given 30, 100, and 300 mg/kg/day, there was an increase in the incidence and severity of periportal hepatocellular vacuolation and increased liver weights in females given 100 and 300 mg/kg/day; based upon the histopathology, the No-Observable Effect Level (NOEL) is 10 mg/kg/day. Slight increases in alanine aminotransferase were noted in the 300 mg/kg/day animals. After the 14 day recovery period, these differences were not noted between the controls and the rats that received 300 mg/kg/day and therefore, based upon the lack of effects after a recovery period, the No-Observable Adverse Effect Level (NOAEL) for the test material in both males and females is 300 mg/kg/day.