Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline confirmed study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: 8,5 weeks
- Weight at study initiation: 240 to 272 grams for males and 184 to 197 grams for females.
- Fasting period before study:
- Housing: suspended stainless steel cages
- Diet (e.g. ad libitum): PMI Certified Rodent Chow, free access
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 14d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: 23.8.-18.10.2000

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly, continuously stirring
- Mixing appropriate amounts with (Type of food): The mixtures were dispensed into amber glass jars daily for dosing.
- Storage temperature of food: RT

VEHICLE PEG 400 and Tween 80 mix
- Justification for use and choice of vehicle (if other than water): not water soluble
- Lot/batch no. (if required): 992286
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Prior to initiation of the 28-day study, homogeneity and stability analyses were performed on concentrations of the test article in the vehicle which bracketed the low- and high-doses administered in this study. Homogeneity analyses were performed on triplicate samples taken from the top, middle and bottom of the two mixtures. Stability of the test article In the vehicle was evaluated on triplicate samples at 0, 5 and 8 days following preparation. Concentration verification analysis was performed on the vehicle and each test article dosing mixture during weeks 1, 2, 3 and 4. The homogeneity, stability and concentration verification analyses were conducted at SLI.
Duration of treatment / exposure:
28d
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Clinical Observations
General health/mortality and moribundity checks were performed twice daily, in the morning and afternoon. A cage-side clinical observation was
performed for each animal daily, between one-half hour and two hours following dosing, and daily during the recovery phase.

Functional Observation Battery (FOB)
a. Home Cage Observations
b. Removal from Home Cage Observations
c. Open Field Observations
d. Manipulative Tests
e. Motor Activity

Bodyweights
Individual bodyweights were recorded on days -2, 8, 15, 22 and 28 (during the treatment phase) and days 35 and 41 (during the recovery phase). In
addition, a terminal body weight was recorded on the day of scheduled euthanasia (day 29 or 42) for calculation of relative organ weight data.

Food Consumption
Food consumption was recorded on days -2, 8, 15, 22 and 28 (during the treatment phase) and days 35 and 41 (during the recovery phase).

Blood was collected from all animals on the day of scheduled euthanasia at the end of the treatment phase (day 29) or the recovery phase (day 42) for
evaluation of selected hematology, coagulation and biochemistry parameters. The blood samples obtained for hematology and biochemistry
evaluations were collected via the orbital plexus while the animals were under light isoflurane anesthesia. The blood samples obtained for the
coagulation evaluations were collected via the vena cava at necropsy. Feed was withheld overnight prior to blood collection, however, water was
available.

Urinalysis
Urine samples were collected from all animals overnight prior to scheduled euthanasia at the end of the treatment phase (day 29) or the recovery phase (day 42). Each rat was housed in a urine collection cage without food, however, the animals were allowed access to water.

Ophthalmology
Ophthalmologlcal examinations were performed on all rats by a board-certified veterinary ophthalmologist. Dr. David A. Wilkie, near the end of the treatment phase (day 23) and the recovery phase (day 40). Eyes were dilated using 0.5% Mydriacyl®ophthalmicsolution priorto biomicroscopic and indirect ophthalmoscopic examination.

Gross Necropsy
All animals were subjected to a complete gross necropsy at the time of death or scheduled euthanasia (day 29 or 42). The necropsy examination included evaluation of the external surfaces of the body and all viscera. At the end of the treatment phase, five males and five females per group were euthanized on day 29. At the end of the recovery phase, the remaining five males and five females in the control and high-dose groups were euthanized on day 42.

Histopathology
All tissues and organs collected at necropsy (days 29 and 42) from all animals in the control and high-dose groups were processed for histopathological examination. The tissues were trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin.
Positive control:
no positive controls

Examinations

Observations and examinations performed and frequency:
behold in details on study design
Sacrifice and pathology:
behold in details on study design
Other examinations:
behold in details on study design
Statistics:
All statistical analyses were performed using a Digital MicroVax 3100 computer. Parametric data was analyzed by one-way analysis of variance (ANOVA). When significance was observed with ANOVA, group by group comparisons were performed using the Tukey-Kramer method. All tests were two-tailed with a minimum significance level of 5%. Rank and count data were analyzed by Kruskal-Wallis Nonparametric ANOVA. When significance was observed, group by group comparisons were performed using Kruskal-Wallis Nonparametric ANOVA. Descriptive (categorical) and quantal data were analyzed by Fischer's
Exact Test. When significance was observed, group by group comparisons were performed using Fischer's Exact Test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
Gross necropsy of the one control male that died on study day 23 revealed a distended and thickened bladder/urethra with a calculi in the bladder. Therefore, this mortality was considered a secondary effect of the bladder calculi which resulted in renal toxicity. This death was not considered treatment-related since this was a control animal and bladder calculi are not uncommon in this species/sex of rat. No remarkable gross necropsy observations were noted for the surviving males or females at the end of the treatment or recovery phases.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion