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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: GLP and guideline conform study, purity of test substance not mentioned (reference to analytical data sheet)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1

Test animals

Species:
rat
Strain:
other: HanBrLWIST (SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd Biotechnology & Animal Breeding Division CH-4414 Fullinsdorf / Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 130-161 grams (mean 143 grams), Females: 112 -133 grams (mean 125 grams)
- Fasting period before study:
- Housing: In groups of five
- Diet: ad libitum
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 5d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES:
Delivery of Animals 14 February 2002
Acclimatization/Pretest 15 to 20 February 2002
Administration/Treatment 21 February to 20 March 2002
Recovery 21 March to 03 April 2002
Termination (Necropsy) 21 March 2002 (Allocation A), 04 April 2002 (Allocation B)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly.
Test item was weighed into a glass beaker on a tared Mettler balance and the
vehicle added (weight:volume). The mixtures were prepared using a magnetic stirrer and
stored at room temperature (17-23°C).
Homogeneity of the test item in the vehicle was maintained during the daily administration
period using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): mixed with vehicle (corn oil)
- Storage temperature of food: RT

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount of vehicle (if gavage): 5 ml(kg bw
- Lot/batch no. (if required): 07939204 and 02938667
- expiry date: 2005 and 2004
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSE FORMULATIONS
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations
were determined in samples taken after experimental start. Concentration and homogeneity
of the dose formulations were determined in samples taken during week 3 of the treatment.
The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics
Division) according to a HPLC method supplied by the Sponsor.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
800 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Male: 10 animals at 0 mg/kg bw/day
Male: 5 animals at 50 mg/kg bw/day
Male: 5 animals at 200 mg/kg bw/day
Male: 10 animals at 800 mg/kg bw/day
Female: 10 animals at 0 mg/kg bw/day
Female: 5 animals at 50 mg/kg bw/day
Female: 5 animals at 200 mg/kg bw/day
Female: 10 animals at 800 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based upon the results of a non-GLP 5-day dose range-
finding study, the test article was administered by
gavage to 2 rats per group and sex. Test item-related
changes were noted in animals treated with 600
mg/kg/day or 1000 mg/kg/day, whereas the findings
noted in the latter group were more clearly defined.
Based on the results of the five-day dose range-finding
study, dose levels of 50, 200 or 800 mg/kg body
weight/day are proposed for this study. In this study,
test item-related findings are expected at 800
mg/kg/day, and 200 mg/kg/day is considered, likely to
be the no-effect level. The highest dose of 1000
mg/kg/day used in the preliminary study was
considered inappropriate for the main study as it was
above the Maximum Tolerated Dose.

- Rationale for animal assignment (if not random): Computer-generated random algorithm
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

FUNCTIONAL OBSERVATION: Yes
- During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks, after 6 weeks
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, 18h
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks, after 6 weeks
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks, after 6 weeks
- Metabolism cages used for collection of urine: Urine was collected during the 18-hour fasting period into a specimen vial.
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4, modified Irwin screen test were evaluated in all animals
- Dose groups that were examined: all
- Battery of functions tested: grip strength, locomotor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
after 4 weeks 21 March 2002 (Allocation A)
after 6 weeks 04 April 2002 (Allocation B)

Adrenal glands
Aorta
Bone (sternum, femur including joint)
Bone marrow (femur)
Brain (cerebrum, cerebellum, brain stem)
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Esophagus
Eyes with optic nerve (fixed in Davidson's
solution)
Harderian gland (fixed in Davidson's solution)
Heart
Ileum, with Peyer's patches
Jejunum with Peyer's patches
Kidneys
Larynx
Lacrimal gland (exorbital)
Liver
Lungs (infused with formalin at necropsy)
Lymph nodes (mesenteric, mandibular)
Mammary gland area
Nasal cavity
Ovaries
Pancreas
Pituitary gland
Prostate gland
Rectum
Salivary glands (mandibular, sublingual)
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord (cervical, midthoracic, lumbar)
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland)
Tongue
Trachea
Urinary bladder (infused with formalin at
necropsy)
Uterus
Vagina
Gross lesions

HISTOPATHOLOGY: Yes,
HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology (following), were processed,
embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with
hematoxylin and eosin.
HISTOPATHOLOGY
Slides of all organs and tissues listed in boldface type (see Necropsy, above) which were
collected at scheduled sacrifice from the animals of control and high-dose groups were
examined by a pathologist.
Other examinations:
ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy:
Brain Thymus
Spleen
Ovaries
Heart
Liver
The organ to terminal body weight ratios as well as organ to brain weight ratios were
determined.
The determination ofthe terminal body weight was performed immediately prior to necropsy.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity,
body weight, organ weights and ratios, as well as clinical laboratory data :
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was
applied if the variables could be assumed to follow a normal distribution for the
comparison ofthe treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test
when the data could not be assumed to follow a normal distribution.
• Student's t-test was applied to grip strength and locomotor activity.
• Fisher's exact-test was applied to the macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All animals survived until scheduled necropsy.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled necropsy.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
FUNCTIONAL OBSERVATIONAL BATTERY

Locomotor Activity
Decreased (statistically significant, p<0.05) mean locomotor activity recorded from 0 to 15
minutes was observed in males and females treated with 800 mg/kg/ when compared with
controls. This was considered to be a possible test item-related effect of uncertain
toxicological relevance.


MICROSCOPIC FINDINGS
The following microscopical changes were noted: In the liver of females a slight diminution of
fatty change at doses of 800 mg/kg/day and in the adrenals of males a slight diminution of
cortical fatty change at doses of 200 and 800 mg/kg/day at the end of treatment. Although
test item-related, these findings are not considered to be of adverse character. After a 14-day
treatment free recovery period, no test item related changes were observed.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Sex:
male/female
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 50 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and 800 mg/kg body weight/day as the no-observed-adverse-effect-level (NOAEL).
Executive summary:

GENERAL

In this subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 800 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 800 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

MORTALITY / VIABILITY All animals survived until scheduled necropsy.

CLINICAL SIGNS No test item related clinical signs of relevance for toxicological evaluation were observed during daily or weekly observations (weeks 1-3).

FUNCTIONAL OBSERVATIONAL BATTERY No test item related findings of relevance for toxicological evaluation were observed during daily or weekly observations (week 4). Grip Strength No test item related differences in fore- or hindlimb grip strength were evident. Locomotor Activity The observed significantly decreased mean locomotor activity in males and females, treated with 800 mg/kg/day, recorded from 0 to 15 minutes was considered to be a test item related effect.

FOOD CONSUMPTION The mean daily and relative food consumption of test item treated animals compared well with those of the control animals.

BODY WEIGHT The mean body weights and mean body weight gain of test item treated animals were comparable with those of the control animals.

CLINICAL LABORATORY INVESTIGATIONS

Hematology No test item related differences in parameters of hematology were evident after four or six weeks when compared with the controls. Clinical Biochemistry No test item related differences in parameters of clinical biochemistry were evident after four or six weeks when compared with the controls. Urinalysis No test item related differences in parameters of urinalysis were evident after four or six weeks when compared with controls.

ORGAN WEIGHTS No test item related differences in mean organ weights, organ to body weight- and organ to brain weight ratios were evident after four or six weeks when compared with the controls.

MACROSCOPIC / MICROSCOPIC FINDINGS At necropsy performed at the end of treatment period and following the recovery period, no test item-related macroscopic findings were observed.

The following histopathological changes were observed: In the liver of females a slight increase of fatty change at doses of 800 mg/kg/day and in the adrenals of males a slight increase of cortical fatty change at doses of 200 and 800 mg/kg/day at the end of treatment. After 14-day treatment-free recovery period, no test item-related changes were observed.