Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2006 - April 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study followed OECD guideline 408 and GLP assurances

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Four minor deviations were noted but in the opinion of the Study Director, these minor deviations did not affect the quality or integrity of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Cyclobutanediol, 2,2,4,4-Tetramethyl (also known as 2,2,4,4-Tetramethyl-1,3-Cyclobutanediol, PM1265
Batch Number: TS060513
Physical Characteristics: Light white powder
Expiration Date: May 17, 2007
Storage: Room temperature
TMC Number: 060439
Purity: 99.79%

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
A total of 90 experimentally naïve CD® [Crl:CD® (SD)] rats/sex (approximately 6 weeks of age) were received from Charles River Laboratories,
Portage, Michigan, on November 8, 2006. During the 14 day acclimation period, the animals were weighed and observed twice daily with respect to
general health and any signs of disease. All animals were given a detailed clinical examination prior to selection for study.

The animals were individually housed in suspended, stainless steel, wire-mesh type cages in an environmentally controlled room. Fluorescent
lighting was provided for approximately 12 hours per day. Temperature and humidity were continuously monitored and recorded. The
protocol-designated ranges were 64 to 79° F and 30 to 70%, respectively.

Meal Lab Diet® (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum, except during designated periods. Tap water
was available ad libitum via an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according
to SOP.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The vehicle and test were administered twice per day (approximately 6 hours apart) for 91 or 92 days during the study via oral gavage. The dose
levels for the treated groups were 25, 80, and 240/200 mg/kg/dose BID at a dose volume of 10 mL/kg/dose BID. On Day 43, the dose level of
240 mg/kg/dose BID was lowered to 200 mg/kg/dose BID. An additional group served as the control and received the vehicle in the same manner and at the same dose level as the treated groups. Individual doses were based on the most recent body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity results show that the batches were accurately prepared with small relative standard deviations between groups. The concentration data show that the weekly test formulations were accurately prepared over the course of the study.
Duration of treatment / exposure:
91 or 92 Days
Frequency of treatment:
Twice daily by oral gavage approximately 6 hours apart
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 160, and 480/400 mg/kg/day
Basis:
other: dosing solutions were analytically verified
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
The animals considered suitable for study were weighed. Using a standard, by weight, block randomization procedure, 80 male and 80 female
animals (weighing 243 to 279 g and 179 to 211 g, respectively, at randomization) were assigned to the control and treatment groups. The dose
levels were selected by the Sponsor, or in consultation with the Sponsor, on the basis of available data from previous studies. Three treatment
groups of 20 male and 20 female CD® [Crl:CD® (SD)] rats were administered the test article at respective dose levels of 25, 80, and 240/200 mg/kg/dose BID (total daily dose of 50, 160, and 480/400 mg/kg/day. The 240 mg/kg/dose BID dose level was lowered to 200 mg/kg/dose BID on Day 43.One additional group of 20 animals/sex served as the control and received the vehicle, distilled water. The test article or vehicle was administered to all groups twice a day (approximately 6 hours apart) for 91 or 92 consecutive days, at a dose volume of 10 mL/kg/dose. Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Observations for clinical signs and masses were
conducted weekly. Functional Observational Battery (FOB) evaulations and locomoter activity monitoring were conducted on designated animals
during Week 13. Detailed neurobehavioral observations were conducted weekly. Body weights were measured and recorded pretest and weekly. Food consumption was measured and recorded weekly. Ophthalmoscopic examinations were conducted pretest and prior to termination. Blood and
urine samples for clinical pathology evaluations were collected from designated animals at termination. At study termination, necropsy examinations were performed, organ weights were recorded, and selected tissues were microscopically examined. Sperm analyses (motility, concentration, and
morphology) were also conducted.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily throughout the duration of the study. On occasion, veterinary consultations were conducted during the course of the study. All treatments and observations were recorded. The medical
treatments and observations are not reported, but are maintained in the study file.

DETAILED CLINICAL OBSERVATIONS:
A detailed clinical examination of each animal was performed weekly. On occasion, clinical observations were recorded at unscheduled intervals. Theobservations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, and nervous system effects including tremors, convulsions, reactivity to handling, and bizarre behavior, and the palpation of masses.

BODY WEIGHT:
Body weights for all animals were measured and recorded 2 days following arrival, prior to randomization on Day -1, and weekly during the study.

FOOD CONSUMPTION:
Food consumption for each animal determined weekly

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic examinations were conducted on all animals by a veterinary ophthalmologist pretest and prior to termination.

CLINICAL CHEMISTRY:
Clinical pathology evaluations were conducted on 10 animals/sex/group at termination (Day 92). The animals had access to drinking water but were fasted overnight prior to sample collection. Blood samples (approximately 4 to 5 mL) were collected via the vena cava after anesthesia with carbon
dioxide inhalation. Samples were collected into tubes containing K3EDTA for evaluation of hematology parameters and citrate for evaluation of
coagulation parameters. No anticoagulant was used for the clinical chemistry samples. The order of bleeding was by alternating one animal from
each dose group, then repeating to reduce handling and time biases.

URINALYSIS:
The animals were housed in stainless steel metabolism cages and urine was collected for at least 16 hours the day before termination.

NEUROBEHAVIOURAL EXAMINATION:
Functional observation battery (FOB) evaluations were conducted without knowledge on the part of the testers of the treatment groups. FOB
evaluations, including those conducted in the home-cage, during handling, in the open field and others, were conducted on all designated animals
(10 animals/sex/group at 0, 25, and 80 mg/kg/dose BID and 8 animals/sex at 240/200 mg/kg/dose BID) at approximately 1 hour following the first dose on one day during Week 13. During open-field evaluations, each animal was observed for a minimum of 3 minutes in a black plexiglass,
observation box measuring 20 x 20 x 8 in. The parameters evaluated in the FOB were based on those outlined in Moser, et al. The observations
included, but were not limited to, evaluation of activity and arousal, posture, rearing, bizarre behavior, clonic and tonic movements, gait, mobility,
stereotypy, righting reflex, response to stimulus (approach, click, tail pinch, and touch), palpebral closure, pupil response, piloerection,
exophthalmus, lacrimation, salivation, and respiration. The amount, qualitative and/or quantitative measures, of defecation and urination were also
recorded. Forelimb and hindlimb grip strength was measured using the procedure described by Meyer, et al., and hindlimb splay was quantitatively measured as described by Edwards and Parker. Pain perception was assessed by measuring the latency of response to a nociceptive (thermal)
stimulus when each animal was placed on a hot plate apparatus set to 52°C as described by Ankier. Body weight and temperature were also measured.

Locomoter activity monitoring was conducted at approximatly 1 hour postdose on one day during Week 13. Testing was conducted on the same
10 animals/sex/group (0, 25, and 80 mg/kg/dose BID) or 8 animals/sex (240/200 mg/kg/BID) desingated for FOB evaulations using
Hamilton-Kinder motor monitoring system. The duration of monitoring was 30 minutes with the data summarized into 10 minute segments. A rangeof different activities were recorded but only the following were used in comparisons between treated and control animals as the most representativeactivity parameters: basic movement, fine movement, rearing, and distance.

A detailed neurobehavioral observation of each animal was performed weekly at 1 to 2 hours following the first dose.An assessment of signs of
autonomic function, general motor activity, gait, posture, neuromuscular function, response to handling, the presence of tonic and clonic
movements, stereotypy, and bizarre behavior. The animal was observed in the home cage for level of activity and scored by the following
scale: (0: quiet or asleep; 1: Alert with routine behavior; 2: Increased movement). The animal was removed from the home cage and placed into a
16 x 16 x 12 in box with 5.3 x 5.3 in grid. The animal was observed for one minute and the number of grids entered with both forefeet were counted and recorded. The animal was initially placed in a corner of the box. While in the grid box the animal was observed for tremors, convulsions,
stereotypy, gait, abnormality, bizarre behavior, and response to a sensory stimulus. Prior to the return of the animal to the cage, the forefeet were
placed on the front of the cage, or on any surface where the animal was able to demonstrate the ability to grip.

SPERM ANALYSIS:
Sperm production was determined following the general procedures outlined by Blazak et al. The right and left testis and epididymis were weighed
separately. The right testis was decapsulated and homogenized, and the homogenate was evaluated for the number of homogenization-resistant
sperm heads using a hemocytometer. A section of the right vas deferens was utilized for videotaping of a prepared sperm sample for automated evaluation of sperm motility (viability) utilizing the Hamilton-Thorne Computer Integrated Visual Optical System (IVOS). The right cauda epididymis was separated, weighed, and used for manual (visual) assessment of sperm concentration. A total of 200 sperm were evaluated for morphology. The
remaining portion of the right epididymis and intact left epididymis were fixed in Bovin’s solution and stored in 10% NBF until processed. Slides were prepared for assessment of sperm morphology from the motility preparations.
Sacrifice and pathology:
Necropsy examinations were performed under procedures approved by a veterinary pathologist on animals found dead and all surviving animals at the scheduled necropsy (Day 92 or 93). Euthanasia was by carbon dioxide inhalation. Euthanasia was confirmed in a manner consistent with SOPs and the carcasses were discarded. The animals were examined carefully for external abnormalities including masses. The skin was reflected
from a ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic, and
cranial cavities were examined for abnormalities and the organs removed, examined, and, where required, placed in fixative. The pituitary was
fixed in situ. All designated tissues were fixed in neutral buffered formalin, except for the eye (including the optic nerve) and testes, which were
fixed using a modified Davidson’s fixative8. Formalin was infused into the lung via the trachea and into the urinary bladder.

Body weights and protocol-designated organ weights were recorded for all surviving animals at the scheduled necropsy and appropriate organ weight ratios were calculated (relative to body and brain weights). Paired organs were weighed together. The thyroid/parathyroid
gland and pituitary gland were weighed following fixation.

Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The slides were examined by a boardcertified veterinary pathologist. A four-step grading system was utilized to define gradable lesions for
comparison between dose groups. The adrenal glands were determined to be target organs and were examined for all groups.

- Adrenal (2)*
- Aorta
- Bone with marrow [femur]
- Bone with marrow [sternum]
- Bone marrow smear [2 collected]a
- Brain [cerebrum, midbrain, cerebellum,
medulla/pons]*
- Epididymis (2)*
- Eye including optic nerve (2)
- Gastrointestinal tract:
esophagus
stomach [glandular and nonglandular]
duodenum
jejunum
ileum
cecum
colon
rectum
- Gonads:
ovary (2)*
testis (2)*
- Gross lesions
- Heart*
- Joint, tibiofemoral
- Kidney (2)*
- Lacrimal gland, exorbital (2)
- Larynx
- Liver [3 sections collected; 2 examined]*
- Lung [collected whole; 2 sections examined]*
- Lymph nodes: mandibular and mesenteric
- Mammary gland [process females only]
- Pancreas
- Peyer’s patch
- Pituitary*
- Prostate* and seminal vesicle (2)
- Salivary gland, mandibular/sublingual [2
collected; 1 examined]#*
- Salivary gland, parotid [2 collected;
1 examined]
- Sciatic nerve
- Skeletal muscle, biceps femoris
- Skin
- Spinal cord [cervical, thoracic, and lumbar]
- Spleen*
- Thymus*
- Thyroid/parathyroid (2)*
- Tongue
- Trachea
- Ureter (2)
- Urinary bladder
- Uterus [both horns]/Cervix*
- Vagina
aBone marrow smears were collected at the scheduled necropsy and held.
#The combined weight of the right mandibular/sublingual salivary gland was obtained.
*Organ weighed
(2) Paired organ
Statistics:
Data for each sex within a set were analyzed separately. Details on the specific type of statistiucal anlaysis are in the below table


Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Test article-related clinical observations were noted only at 80 and 240/200 mg/kg/dose BID. At 80 mg/kg/dose BID, clonic convulsions were
seen in 1 male in Week 11, 2 males in Week 13, and 1 female in Week 10. Hypersensitive to touch was seen in the females and stereotypic behavior was seen in both genders at 80 mg/kg/dose BID. At 240/200 mg/kg/dose BID, in addition to clonic convulsions (noted in Weeks 2, 3, 6, and 7),
hypersensitive to touch was seen in both genders as well as stereotypic behavior. The stereotypy was most frequently characterized as jumping and rigidity; the rigidity could have also been another convulsion. The highest incidences of convulsions and stereotypy were seen in the first few weeks. The convulsions were only observed shortly after dosing and were transient in nature.

There were 11 male and 13 female early deaths at 240/200 mg/kg/dose BID and 1 female at 80 mg/kg/dose BID. A cause of death was
undetermined in all females and most males. Cause of death in two males from the 240/200 mg/kg/dose BID group was aspiration of foreign
material (animal number 1062) and urogenital/inflammation/obstruction/calculi (animal number 1071).

URINALYSIS
There was a slightly decreased urine pH in both males (10%) and females (3%) at 240/200 mg/kg/dose BID with statistical significance present only in males. In addition, males at 240/200 mg/kg/dose BID had significantly increased urine volumes (1.95 fold) versus controls. These urine volume changes in males were potentially not toxicologically relevant due to the relatively small magnitude of increase, and urine volumes often have a
large degree of expected variability, not necessarily associated with any treatment.

NEUROBEHAVIOUR
In the weekly neurobehavioral observations 1 to 2 hours postdose, the convulsions described above were seen in a few 240/200 mg/kg/dose BID
animals. In addition, at 240/200 mg/kg/dose BID the grid activity count data for the males showed increased activity.

At the end of the 13-week study there was no test article-related effect on motor activity.

In the FOB evaluations there were clearly no dose- or time-dependent changes in any other sensory (hot plate, righting reflex), motor (open field, hindlimb splay, general activity), autonomic (reflexes), or central nervous system domain of neurological function in these rats. The range of grip
strength values demonstrated across treatment groups was well within the normal within- and between-laboratory variations reported by the
International Programme on Chemical Safety (IPCS) within the World Health Organization (WHO) and by Ross et al.

ORGAN WEIGHTS
Statistically significantly increased adrenal gland weights were present in males and females at 240/200 mg/kg/dose BID (all parameters) and in
males at 80 mg/kg/dose BID (absolute and relative to brain weight) when compared to controls. Mean absolute adrenal gland weights were increased19% and 41% in males at 80 and 240/200 mg/kg/dose BID, respectively and 33% in females at 240/200 mg/kg/dose BID. Statistically significantly
increased mean kidney weights were present in males at 25, 80, and 240/200 mg/kg/dose BID when compared to controls. Mean absolute kidney
weights were increased 7%, 12%, and 18% in males at 25, 80, and 240/200 mg/kg/dose BID, respectively. There was no microscopic correlate for the increased weights in kidneys examined and the percent of weight increase was considered not to be biologically significant.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test article-related microscopic changes were present in the adrenal glands of males and females at 240/200 mg/kg/dose BID and in males at
80 mg/kg/dose BID. Diffuse cortical hypertrophy/hyperplasia was present in 5 and 8 males at 80 and 240/200 mg/kg/dose BID, respectively and in 2 females at 240/200 mg/kg/dose BID. Diffuse cortical hypertrophy/hyperplasia was characterized by a minimal to mild increase in the size
and number of cells in the zonas fasciculata and reticularis of the adrenal cortex.

OTHER FINDINGS: SPERM ANALYSIS
At 80 mg/kg/dose BID, the sperm concentration per cauda epididymis was significantly reduced. However, this was considered to be sporadic in nature and not test article-related because other sperm parameters, including the sperm concentration per gram cauda epididymis, were not affected.
Both the total sperm concentration per cauda epdidymis and concentration per gram cauda epididymis endpoints are inter-dependent; i.e., a
significant reduction one of them will lead to the same effect on the other.

At 240/200 mg/kg/dose BID, sperm motility was decreased, but not significantly. This decrease in percent motile sperm was mainly due to one male rat (animal number 1064) which had sperm motility of 0%. Total sperm concentration per cauda epididymis and sperm concentration per gram cauda epididymis were significantly reduced at 240/200 mg/kg/day when compared to the control animals. The sperm concentrations for animal 1064
were much lower than the average sperm concentrations in the group. This finding was consistent with the microscopic findings of the epididymis
(oligospermia/germ or cell debris) in this animal. However; even if the data of this animal were removed from the mean data, the total sperm
concentration per cauda epididymis and sperm concentration per gram cauda epididymis will be still decreased; thus, the decrease in these two
sperm parameters were considered to be test article-related. In addition, the group mean percent abnormal sperm was significantly increased at 240/200 dose group. However, the increase in percent of sperm morphology was again due to animal number 1064. This male rat was found to have 99.5% of abnormal sperm. If the data of this animal were removed from the mean value, the rest of the group will have a mean percent of abnormal
sperm of (4.125 ± 1.408), which is comparable to the values in the historical control. Therefore, the increase of abnormal sperm at
240/200 mg/kg/dose BID was not considered to be test article-related.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
50
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effects were noted at this dose level.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
50
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, where rats were dosed orally for 13-weeks at 25, 80,
240/200 mg/kg/dose BID (50, 160, and 480/400 mg/kg/day) the dose level of 50 mg/kg/day was considered to be a NOAEL
(no-observed-adverse-effect-level).
Executive summary:

Three treatment groups (20/sex) of rats were administered the test article at dose levels of 25, 80, and 240/200 mg/kg BID (50, 160 and 480/400 mg/kg/day). Controls received distilled water vehicle. The 240 mg/kg/dose BID dose level was lowered to 200 mg/kg/dose BID on Day 43 due to increased mortality.

In this study, no test article-related adverse effects were seen on body weight, hematology, coagulation, clinical chemistries, urinalysis, or macroscopic evaluations.

There were 11 male and 13 female early deaths at 240/200 mg/kg BID and 1 female at 80 mg/kg BID. A cause of death was undetermined in all females and most males. Cause of death in two males from the 240/200 mg/kg BID group was aspiration of foreign material in one and urogenital/inflammation/obstruction/calculi in the other.

Test article-related clinical observations were noted at 80 and 240/200 mg/kg BID. At 80 mg/kg BID clonic convulsions were seen in 1 male in Week 11, 2 males in Week 13, and 1 female in Week 10. Hypersensitive to touch was seen in the females and

stereotypic behavior was seen in both genders at 80 mg/kg BID. At 240/200 mg/kg BID, in addition to clonic convulsions (noted in Weeks 2, 3, 6, and 7), hypersensitive to touch was seen in both genders as well as stereotypic behavior. The stereotypy was most frequently characterized as jumping and rigidity; the rigidity could have also been another convulsion. The highest incidences of convulsions and stereotypy were seen in the first few weeks. The subsequent lower incidence was attributed to lowering the dose level on Day 43. At the end of the 13 -week study, there were no test article-related effects seen in the functional observational battery of tests or motor activity.

In the weekly neurobehavioral observations 1 to 2 hours postdose, the convulsions described above were seen in a few 240/200 mg/kg BID animals. In addition, at 240/200 mg/kg BID the grid activity count data for the males showed increased activity.

However, at the end of the 13 -week study there was no test article-related effect on motor activity. The possible differences that were noted were sporadic with no relationship to dose.

There was a transient test article-related decrease in food consumption for the first week in both the males and females at 240/200 mg/kg BID. All groups consumed a similar amount of food each week after that. Test article-related organ weight changes were present in adrenal glands. Statistically significantly increased mean kidney weights were present in males at 25, 80, and

240/200 mg/kg/dose BID when compared to controls. Mean absolute kidney weights were increased 7%, 12%, and 18% in males at 25, 80, and 240/200 mg/kg/dose BID, respectively. There was no microscopic correlate for the increased weights in kidneys examined and the percent of weight increase was considered not to be biologically significant. Statistically significantly increased adrenal gland weights were present in males and females at 240/200 mg/kg BID (all parameters) and in males at 80 mg/kg BID (absolute and relative to brain weight) when compared to controls. Mean absolute adrenal gland weights were increased 19% and 41% in males at 80 and 240/200 mg/kg BID, respectively and 33% in females at 240/200 mg/kg BID. The increased adrenal gland weights correlated microscopically to diffuse cortical hypertrophy/hyperplasia. Diffuse cortical hypertrophy/hyperplasia was present in 5 and 8 males at 80 and 240/200 mg/kg BID, respectively and in 2 females at 240/200 mg/kg BID. Diffuse cortical hypertrophy/hyperplasia was characterized by a minimal to mild increase in the size and number of cells in the zonas fasciculata and reticularis of the adrenal cortex. This was the only microscopic finding in the study despite the neurobehavioral observations.

Test article-related sperm effect was limited to the significant reduction in total sperm concentration per cauda epididymis and sperm concentration per gram cauda epididymis at 240/200 mg/kg/dose BID. This was likely a stress related response.

In conclusion, under the conditions of this study, where rats were dosed orally for 13 -weeks at 25, 80, 240/200 mg/kg/dose BID, the 25 mg/kg/dose BID level (50 mg/kg/day) was considered to be a NOAEL (no-observed-adverse-effect-level).