Dimethyl 2,2'-azobis(2-methylpropionate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 25 1997 to February 26 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl 2,2'-azobis(2-methylpropionate)

EC Number:

219-976-6

EC Name:

Dimethyl 2,2'-azobis(2-methylpropionate)

Cas Number:

2589-57-3

Molecular formula:

C10H18N2O4

IUPAC Name:

dimethyl 2,2'-azobis(2-methylpropionate)

impurity 1

Reference substance name:

unknown

Molecular formula:

unknown

IUPAC Name:

unknown

Test material form:

solid: crystalline

Specific details on test material used for the study:

Name of Substance in Test report: V601

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: KCQ 7587
- Expiration date of the lot/batch: not stated
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigeration (- 4°C) in the dark
- Stability under test conditions: The test article was considered stable when stored refrigerated.
- Solubility and stability of the test substance in the solvent/vehicle: not stated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Dose formulations of V-601 in corn oil were prepared weekly by direct dilution by mixing
appropriate amounts of corn oil with test article using a stir plate. The dose formulations were stored
at room temperature prior to use.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

according to Guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc., St. Constant, Quebec
- Females (if applicable) nulliparous and non-pregnant: not stated
- Age at study initiation: 49 to 50 days
- Weight at study initiation: 238 to 30I g (males) or I55 to 200 g (females).
- Fasting period before study: no
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2.5 to 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
A standard certified commercial pelleted laboratory diet (PMI Certified Rodent Chow 5002) was provided ad libitum except during designated procedures. On occasion, it was necessary to give certain animals the same food only in a powdered form. The diet was controlled and routinely analyzed by the manufacturer for maximum allowable concentrations of contaminants (e.g., heavy metals, atlatoxins, organophosphates, chlorinated hydrocarbons and PCBs ).
Water which had been further treated by reverse osmosis and ultraviolet sterilization was provided ad libitum except during designated procedures. Periodic analysis of the water was subcontracted to management authorized analytical laboratories which were audited by the Quality Assurance Department ofClinTrials BioResearch Ltd. The results of these analyses are retained in the scientific archives of ClinTrials BioResearch Ltd.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%,
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): I2 hours light and I2 hours dark

IN-LIFE DATES: From: November 7, 1997 To: February 26, 1998

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Dose formulations of V-601 in corn oil were prepared weekly by direct dilution by mixing
appropriate amounts of corn oil with test article using a stir plate. The dose formulations were stored at room temperature prior to use.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility reasons
Identity Corn oil
Batch/Lot No. 86H0059
Expiry Date · November/December 2002
Description Yellow liquid
Storage Conditions Room temperature in the dark
Supplier Sigma Chemical Co.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to treatment, the homogeneity and stability of the lowest dose formulation were determined.
The results indicated that the dose formulation was homogeneous and stable for up to 10 days at
room temperature. However, because the results were slightly low (average recovery 88%) for the
homogeneity assessment (attributed to the analytical chemistry method), the method of analysis was
changed and a re-analysis for homogeneity and stability (up to 14 days at room temperature) was
incorporated into the week 1 dose formulation preparation for the lowest dose group. Homogeneity
and stability assessments to cover the higher dose levels were evaluated on a previous study (CTBR
Project Number 97454).
Samples of the dose formulations used for administration to the animals during weeks 1 to 4, 8 and
13 were analyzed for concentration of the test article.
Prior to analysis oftest samples generated during the study, the analytical method was validated at
ClinTrials BioResearch Ltd.
Test article concentrations were considered acceptable when the mean of the individual samples
analyzed was within ± 10% of the nominal concentration. Such analyses were performed by
ClinTrials BioResearch Ltd~

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

3 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on range finding tests
- Rationale for animal assignment (if not random): At least one week before treatment initiation, all animals were weighed and 15 males and 15 females were assigned to each of 4 groups using a computer-based selection procedure which ensured the homogeneity of group means and variances for body weight. Males and females were assigned separately.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

Clinical Examinations
All animals were examined twice daily for mortality and signs of ill health or reaction to treatment.
A check for the presence of salivation was conducted before and after dosing each day during the
treatment period. In addition, a complete detailed examination was performed weekly commencing
one week prior to treatment initiation.
Any observed clinical signs were individually recorded.
4.2 Body Weight
The animals were weighed at least twice prestudy (days -7 and -6 only reported) and then weekly
during the dosing period including days of behavioral testing.
4.3 Food Consumption
Individual food consumption was measured weekly commencing the last week of the acclimation
period. ·
4.4 Functional Observational Battezy (FOB) ·
All animals were examined once prior to treatment initiation (prestudy) and once during the 4th, 8th
and l 3th weeks of treatment. The procedure for the qualitative examination is shown in the SOP
(Appendix 4).
Testing was performed by the same trained technicians, where possible, who were unaware of the
animals treatment.
The FOB was performed with equipment built for this purpose. The arena was a 2' square of
plexiglass placed on a raised surface. The tests were conducted in the room housing the animals
where temperature, humidity and photoperiod are controlled. Odors in the room were minimized by
maintaining adequate air changes and cleaning of equipment, as necessary.
Qualitative
Observations in Home Cage
body position
tremors, twitches, convulsions
bizarre/stereotypic behavior
Removal from Home Cage
ease of removal
vocalization
Observations in Arena
reanng
ataxic, hypotonic and impaired gait
overall gait incapacity
bizarre/stereotypic behavior
palpebral closure
tremors, twitches, convulsions
piloerection
respiratory rate/pattern
locomotor activity level
arousal
groom mg
defecation
urination
olfactory response
Handling Observations
lacrimation
pupil size
salivation
urinary staining
diarrhea
body tone
extensor thrust
corneal reflex
pinna reflex
toe and tail pinch
visual placing
On Surface
auricular startle
air righting reflex
On Top of Box
positional passivity
4.4.2 Quantitative
4.4.2.1 Grip Strength
Calibration
Before the start of testing and following completion of testing on each day, the Chatillon strain
gauges were checked using calibration weights and the readings recorded.
Forelimb
The dial on the gauge was set to "O". The rat was held by the body and/or tail and allowed to grip
the mesh, and then was pulled slowly and steadily until it released its grip. Maximum strain was
recorded two times alternating with hindlimb grip testing.
Hindlimb
The dial on the gauge was set to "0". The rat was allowed to set its hindpaws against the mesh and
was pulled backward by the base of the tail until it released its grip. The maximum strain was
recorded two times alternating with forelimb grip testing.
4.4.2.2 Hindlimb Splay
Landing foot spread was measured using inking of the hind feet. Hindlimb splay was recorded
twice. The ink was wiped off the feet after testing using a paper towel dampened with water.
4.4.2.3 Body Temperature
The rectal probe was gently inserted, the reading allowed to stabilize and the temperature then
recorded. ·
4.5 Motor Activity
Fallowing the FOB assessment, the animals were transferred to a testing room where activity levels
were measured individually in figure 8 enclosures. All animals were tested prior to treatment
initiation (prestudy) and once during the 4th, 8th and 13th weeks of treatment. Animals from the
control and treated groups were balanced across enclosures, where possible, using a preassigned
distribution. The sessions were of I-hour duration and activity counts were recorded by a
microcomputer in 6 successive I 0-minute intervals.
In the testing room, temperature and humidity were monitored, and a background sound level of
approximately 70 dBA and an illumination of approximately 800-1200 Lux maintained throughout
testing. Light levels in the testing room were measured before the start of testing and following
completion of testing on each day. The sound level was recorded on a continuous basis throughout
testing on each day.
In addition to the "diagnostic" function in the system, a check of each beam was made by manually
"breaking" each beam a predetermined number of times and verifying that the "breaks" were
properly recorded. These checks were made at least prior to the start of testing and at the completion
of testing on each day.
4.6. Ophthalmology
Once prior to the start of treatment (prestudy) and again during week 13 of treatment (before
dosing), all animals were subjected to funduscopic (indirect ophthalmoscopy) and biomicroscopic
(slit lamp) examinations. The mydriatic used was 0.5% atropine sulfate. Examinations were
performed by a board certified veterinary ophthalmologist.
4. 7 Laboratory Investigations
Prior to commencement of treatment, laboratory investigations (hematology and biochemistry) were
performed on the I 0 male and I 0 female health screen animals. Blood samples were obtained from
the abdominal aorta following isoflurane anesthesia.
These same investigations were performed on all animals during week 5 and on all animals except
those perfused at study termination.
During week 5, the animals were bled from the tail vein. At study termination, blood samples were
collected from the abdominal aorta.
Food was removed overnight from animals prior to blood sampling.
Information on the procedures used for the laboratory investigations and the units and symbols used
to report the data are included in Appendix 8.
4.7.1. Hematology (using EDTA or Citrate* as anticoagulant)
Parameters examined:
* activated partial thromboplastin time
blood cell morphology
- 18 -
PROJECT NO. 97455
erythrocyte indices (mean corpuscular volume - MCV; mean corpuscular hemoglobin - MCH;
*
mean corpuscular hemoglobin concentration - MCHC; and red cell distribution width - RDW)
hematocrit
hemoglobin
mean platelet volume
platelet count
prothrombin time
red blood cell count
reticulocyte count
white blood cell count (total, absolute and percent differential)
Bone marrow smears were prepared as described in TERMINAL PROCEDURES Section 5 .3 Tissue
Preservation but were not examined.
4. 7 .2. Clinical Biochemistry
Parameters examined:
A/G ratio (calculated)
alanine aminotransferase
albumin
alkaline phosphatase
aspartate aminotransferase
blood urea nitrogen
calcium
chloride
cholesterol
creatinine
globulin (calculated)
glucose
inorganic phosphorus
protein electrophoresis
potassium
sodium
total bilirubin
total protein
triglycerides

Sacrifice and pathology:

5 .1 Gross Pathology - Animals not selected for Perfusion
Prior to the commencement of treatment, 10 male and 10 female health screen animals were
euthanized by exsanguination from the abdominal aorta following isoflurane anesthesia. These
animals were subjected to external and internal gross examinations. Tissues were not retained.
All animals found dead during the study were subjected to necropsy and tissue samples were
preserved. Prior to necropsy, the carcass was stored at circa 4°C.
Ten animals/sex/group (maximum) euthanized on completion of the treatment period were
euthanized by exsanguination from the abdominal aorta following isoflurane anesthesia and blood
sample collection. In order to avoid autolytic change, a complete gross pathology examination of
the carcass was conducted immediately on all animals which were euthanized. All animals were
fasted overnight before scheduled euthansia. Terminal body weight was recorded at necropsy.
All necropsies were conducted under the supervision of a pathologist and necropsy consisted of an
external examination, including identification of all clinically recorded lesions, as well as a detailed
internal examination.
5 .2 Organ Weight Assessment - Animals not selected for Perfusion
For each animal euthanized at completion of treatment, the following organs were dissected free of
fat and weighed:
adrenal glands
brain
heart
kidneys
liver
lungs
ovaries/testes
pituitary
prostate
spleen
thymus
thyroid. lobes and parathyroid glands
uterus
Paired organs were weighed together.
Organ weight ratios relative to body weights were calculated.
5 .3 Tissue Preservation - Animals not selected for Perfusion
On completion of the necropsy of each animal, the following tissues and organs were retained.
Neutral buffered 10% formalin was used for fixation and preservation unless otherwise indicated:
abnormalities
a animal identification
adrenals
aorta (thoracic)
**bone and marrow (sternum)
brain (cerebrum, cerebellum, midbrain and medulla oblongata)
cecum
cervix
colon
duodenum
*epididymides
esophagus
*eyes
Harderian glands
heart (including section of aorta)
ileum
jejunum
kidneys
lacrimal glands
liver (sample of 2 lobes)
++lungs (all lobes)
lymph nodes (mandibular and mesenteric)
+mammary gland (inguinal)
*+ optic nerves
ovaries
pancreas
pituitary
prostate
rectum
salivary gland
sciatic nerve
seminal vesicles
skeletal muscle
skin (inguinal)
spinal cord (cervical)
spleen
stomach
*testes
+thymus+ thyroid lobes (and parathyroids)
tongue
trachea
urinary bladder
uterine horns
uterus
vagina

* Fixed in Zenker's fluid (euthanized animals only).
* * Bone decalcified prior to sectioning.
+ Examined histopathologically only if present in routine sections of eyes (optic nerves), thyroid
lobes (parathyroid glands), or skin (mammary gland).
++ Infused with neutral buffered 10% formalin. ( euthanized animals only)
a Retained but not processed.
For all euthanized animals, 3 femoral bone marrow smears were prepared, one of which was stained
with May-Grlinwald-Giemsa. The smears were retained but not evaluated.
5 .4 Histopathology - Animals not selected for Perfusion
All tissues from animals in the control and high dose groups (10/sex/group) were prepared for
histopathological examination by embedding in paraffin wax, sectioning and staining with
hematoxylin and eosin and examined as follows:
Groups 1 and 4:
Groups 2 and 3:
Tissues_ listed under tissue preservation
All gross lesions
5 .5 Tissue Preservation - Animals Selected for Perfusion
Following completion of treatment, 5 rats/sex/group, randomly selected, were deeply anesthetized
by an intraperitoneal injection of sodium pentobarbital (approximately 35 mg/kg or as required).
When anesthesia was deep, the thorax was opened. A 16-gauge needle was inserted into the left
ventricle and the right atrium was opened. Perfusion with lactated Ringer's solution containing
heparin (1000 IU/liter) and sodium nitrite (0.02 g/L) was initiated and continued until the auricular
effluent was essentially free of blood. The perfusion fluid was then changed to a mixture of 3%
glutaraldehyde, 3% paraformaldehyde, 0.05% calcium chloride and 0.1 % picric acid in 0.1 M
cacodylate buffer (pH 7.3 to 7.5).
5.6 Neuropathology - Animals Selected for Perfusion
On completion of the perfusion, the calvarium and the dorsal vertebral column were exposed by
removing the skin and underlying muscle. The skin on the lateral surface of the hindlimbs was also
removed. The skin and tissues on the ventral surface of the abdomen and thorax was removed and
discarded. The thoracic and abdominal organs were removed as two groups of tissues and placed
in a separate tissue bag in neutral buffered 10% formalin but were not examined. The remaining
carcass containing the brain, spinal cord and limbs were placed in another tissue bag containing
neutral buffered 10% formalin.
Tissues from the high dose and control animals euthanized after 13 weeks of treatment were
processed for neuropathological evaluation. The trimmed tissues from the low and mid dose groups
were kept in neutral buffered 10% formalin but were not examined. The remaining carcasses from
all animals were retained but not examined. The nervous system tissues of animals in all groups
were grossly examined at the time of sampling and any gross findings were recorded and reported.
5 .6.1. Tissues for Paraffin Embedding. Sectioning and Staining
The tissues listed below from the high dose and control animals euthanized after 13 weeks of
treatment were prepared for examination by embedding in paraffin wax and sectioning at 6 microns.
Adjacent sections of brain and spinal cord were stained with hematoxylin and eosin and then
examined by light microscopy.
Brain (6 levels) - forebrain (through the septum), center of the cerebrum (through the
hypothalamus), midbrain, cerebellum and pons, midcerebellum and medulla oblongata, and medulla
oblongata.
Spinal cord - cervical and lumbar (cross-section)
Grossly abnormal central nervous system tissues
The skeletal muscle (gastrocnemius) and thoracic spinal cord were retained but not examined.
Brain weight (excluding olfactory bulbs), length and maximum coronal width were recorded prior
to trimming.
5.6.2. Tissues for Epoxy Embedding. Sectioning and Staining
The tissues listed below, from the control and high dose group animals, euthanized after 13 weeks
of treatment were rinsed in 0.1 M sodium cacodylate buffer and placed in 2% osmium tetroxide for
2 hours. Each piece of tissue was rinsed in buffer and stained in a 1 % aqueous solution of uranyl
acetate for 2 hours. The tissues were then rinsed in distilled water, dehydrated in ascending concentrations
of ethyl alcohol and embedded in a mixture of Jembed and Araldite. Epoxy sections
(0.5 μm) were obtained with a glass knife, stained with borate-buffered 1 % toluidine blue, cover
slipped and examined by light microscopy.
Peripheral Nervous System (PNS)
Sciatic nerve (mid-thigh region) (cross-section)
Sciatic nerve (at sciatic notch) (cross-section)
Sural nerve (at knee) (cross-section)
Tibial nerve (at knee) (cross-sections)
Central Nervous System (CNS)
Gasserian ganglion - left (cross-section); right used for animal 4013
Lumbar dorsal root ganglion (L4) (cross-section)
Lumbar dorsal root (L4) (cross-section)
Lumbar ventral root (L4) (cross-section)
Cervical dorsal root ganglion (CS) (cross-section)
Cervical dorsal root (CS) (cross-section)
Cervical ventral root (CS) (cross-section)
Grossly abnormal central or peripheral nervous system tissues.
The tibial branches to the calf musculature were retained but not examined.

Statistics:

Group variances for body weight, food consumption, laboratory investigations, quantitative FOB (count data was transformed before analysis) and organ measurement data were compared using Bartlett's test. When the differences between group variances were not significant (P~0.001), a one-way analysis of variance (ANOVA) was performed. If significant differences (P[continued in any other information on materials and methods incl. tables]

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no overt clinical signs seen for the treated groups.
The most frequent clinical finding was salivation which occurred post-dosing for males and females
in both control and treated groups. In general, this finding was seen during the first few days of
dosing for the control and all treated groups, but thereafter occurred mostly for animals in the 10
mg/kg/day group and to a lesser extent for some animals in the 3 mg/kg/day group. Salivation was
also seen occasionally pre-dosing but only for animals in the 3 and 10 mg/kg/day groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One 10 mg/kg/day male (no. 4007) was found dead during week 12 (day 82). No abnormal clinical
signs were observed just prior to death. Red material on the tray under the rat's cage was noted at
the observation of death. This mortality was likely unrelated to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant differences between the control and treated groups.
Slightly lower average body weights were observed for males in the 1 and 3 mg/kg/day groups
generally during the second half of the treatment period. However, as no comparable effect was seen
for 10 mg/kg/day males, this was attributed to intergroup variability.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant differences in food intake that were related to treatment.
On one occa,sion for the 10 mg/kg/day males and three occasions for 3 mg/kg/day males, the food
intake was statistically significantly different from the control group. However, as no comparable
change was detected for the 10 mg/kg/day females, these differences were attributed to intergroup
variabilty.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular findings that were related to treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the week 5 assessment, the 10 mg/kg/day males showed a significant (P<0.05) increase in
absolute/relative monocytes and RDW and significant (P<0.05, P<0.01 or P<0.001) decreases in
MCV, MCH and MCHC. Females in the 10 mg/kg/day group showed significant (P<0.05 or
P<0.01) decreases hemoglobin and hematocrit as well as for MCV and MCH, and a significant
(P<0.05) increase for RDW. The white blood cell count was significantly (P<0.05) decreased for the
3 mg/kg/day females.
At the week 14 assessment, hemoglobin and hematocrit as well as MCV and MCH were
significantly (P<0.05 or P<0.01) decreased, and RDW was significantly (P<0.05 or P<0.01)
increased for males and females in the 10 mg/kg/day group.
It is possible that the hematological changes seen for the 10 mg/kg/day group may have been the
result of treatment with V-601, however, in the absence of pathological lesions attributed to
administration with V-601, these changes were considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the week 5 assessment, 10 mg/kg/day males showed a significant (P<0.05 or P<0.01) increase
in glucose and chloride, and alanine aminotransferase (ALT) levels were significantly (P<0.01)
decreased for males and females in this group. A significant (P<0.05 or P<0.01) increase in
phosphorus level and relative beta globulin (electrophoresis) were also seen for 10 mg/kg/day
females, as were significant (P<0.01) decreases for absolute and relative alpha globulin
(electrophoresis). Males in the 3 mg/kg/day group showed a significant (P<0.05) increase in glucose,
and males and females in this group had a significant (P<0.01) decrease in ALT. Females in the 3
mg/kg/day group also showed significant (P<0.05 or P<0.01) increases in relative beta globulin
(electrophoresis) and decreases in absolute and relative alpha globulin (electrophoresis). Females
in the 1 mg/kg/day group showed a significant (P<0.05) increase in chloride levels and a significant
(P<0.01) decrease in absolute and relative alpha globulin (electrophoresis).
At the week 14 assessment, creatinine was significantly (PALT was significantly (P<0.01 or P<0.001) decreased for 10 mg/kg/day males and females, total
protein was significantly (P<0.05) decreased for females in the 10 mg/kg/day group, and potassium
was significantly (P<0.05) increased for 10 mg/kg/day females. Creatinine was also significantly
(P<0.05) decreased for 3 mg/kg/day males, and ALT was significantly (P<0.05 or P<0.01) decreased
for males and females in this group. Creatinine was significantly (P<0.05) decreased for 1
mg/kg/day males, and females in this group showed significant (P<0.05 or P<0.01) decreases in total
protein, globulin and calcium.
In the absence of any treatment-related pathological findings, the above changes were considered
to be not toxicologically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional Observational Battery (FOB)
There were no marked differences detected between the control and treated groups that indicated an
effect of treatment.
Significant (P<0.05 or P<0.01) decreases in defecation for 1and3 mg/kg/day males at the week 13
assessment and an increase in locomotor activity for the 1 mg/kg/day females at the week 8
assessment occurred. However, as no similar effects were observed for the 10 mg/kg/day group,
these differences were attributed to intergroup variability.

Motor Activity
There were no clear treatment-related effects.
Males in the l and 10 mg/kg/day groups showed a significant (P<0.05 or P<0.01) increase in total
counts at the week 13 assessment when compared to the control group. This was considered unlikely
to be related to treatment for the following reasons: no dose-response relationship was seen, there
was no significant change on prior testing occasions, there were no effects detected using the FOB,
and no comparable effect was observed for treated females. The statistitical significance was
considered likely attributable to a lower control group value.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

For non-perfused animals, the absolute and relative (to body weight) thymus weight of females in
the 10 mg/kg/day group were significantly (P < 0.05) decreased. In addition, the absolute spleen
weight of 3 mg/kg/day males was significantly (P<0.01) decreased and the relative gonad weight
of 1 mg/kg/day males was significantly (P<0.05) increased. In the absence of any pathological
findings, these changes were considered not to be of toxicological significance.
No significant differences between the control and treated groups were detected for brain
measurements of perfused animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Animal 4007 treated at 10 mg/kg/day that died showed a clot in the oral cavity and multiple dark
areas in the mucosa of the glandular portion of the stomach. No cause of death was established.
No findings were seen for perfused or non-perfused animals euthanized at study completion that
were clearly attributed to treatment.

Neuropathological findings:

no effects observed

Description (incidence and severity):

see Histopathological findings: non-neoplastic

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Animal 4007 treated at 10 mg/kg/day showed hemorrhage in the lung and no other findings. No
cause of death was established.
No findings were seen for perfused or non-perfused animals euthanized at study completion that
were clearly attributed to treatment.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

see Histopathological findings: non-neoplastic

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

neuropathology

ophthalmological examination

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Treatment of male and female rats with V-601 for 13 weeks at dose levels of up to 10 mg/kg/day
did not result in toxicity (including neurotoxicity).

Executive summary:

Experimental Procedures

Groups of 15 male and 15 female Sprague-Dawley rats were treated for 13 weeks, by gavage, with

V-601 at dose levels of 1, 3 or 10 mg/kg/day in order to evaluate its potential toxicity. A control

group of the same size was handled in an identical manner except that it received the vehicle only,

corn oil, at the dose volume for the study of 5 ml/kg/day. Body weights and food intake were

measured weekly, abnormal clinical signs were recorded each day, and an ophthalmological

examination was performed prior to treatment initiation and during week 13. In addition, a

functional observational battery (FOB) and motor activity test were performed prior to treatment

initiation and then during weeks 4, 8 and 13 (pre-dosing). Blood samples for hematological and

biochemistry evaluations were collected during week 5 and at study completion (non-perfused

animals only). Following completion of the treatment period, 5 rats/sex/group were perfused and

these animals, in the control and high dose groups, underwent a histopathological examination of

central and peripheral nervous system tissues. The remaining animals (up to 10/sex/group) were also

euthanized at treatment termination and were subjected to necropsy with organ weights recorded and

tissues retained. Tissues from these animals in the control and high dose groups were then given a

histopathological examination.

Results

No animals died due to treatment. One animal (10 mg/kg/day male) was found dead during week

12.

There were no overt clinical signs seen for the treated groups. The most frequent clinical finding was

salivation which occurred post-dosing for males and females in both control and treated groups. In

general, this finding was seen during the first few days of dosing for the control and all treated

groups, but thereafter occurred mostly for animals in the 10 mg/kg/day group and to a lesser extent

for some animals in the 3 mg/kg/day group. Salivation was also seen occasionally pre-dosing but

only for animals in the 3 and 10 mg/kg/day groups.

The body weights and food consumption were similar between the control and treated groups.

There were no behavioral changes (based on FOB and motor activity evaluations) considered related

to treatment.

No ophthalmological effects were observed.

There were no gross or histopathological findings (nervous and non-nervous system tissue) or organ

weight changes that indicated an effect due to treatment.

Laboratory investigations occasionally indicated significant effects on hematological and blood

biochemical parameters but in the absence of any pathological change they were considered not to

be of toxicological significance.