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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Nov 1997 - 16 Mar 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
ToxLabs Prüflabor GmbH, Greppin, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: 32-38 d
- Weight at study initiation: 148.5 g (mean value males), 136.7 g (mean value females)
- Housing: one or two animals in cages (Makrolon Type 3)
- Diet: Altromin 1326, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6-8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 30-60
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: distilled water containing 1% Tween 80
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 1%
- Lot/batch no. (if required): S23350 739
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 2 mL samples of each formulation were taken and stored in the frozen state until measurement
Duration of treatment / exposure:
90 d
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 (control, test and satellite groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for selecting satellite groups: 10 animals each from the high dose and the vehicle group were used to investigate reversibility of possible effects
- Post-exposure recovery period in satellite groups: 28 d
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, autonomic activity, presence of clonic or tonic movements, stereotypies, bizarre behavior
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes, changes in skin, fur, eyes, mucous membranes, gait, posture: response to handling; occurrence of secretions and excretions
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly from the start to the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the administration and at the end of the study
- Dose groups that were examined: All (surviving) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study
- Anesthetic used for blood collection: Yes (Ether)
- Animals fasted: Yes, over night
- How many animals: All animals
- Parameters examined: erythrocyte count, hemoglobin concentration, packed cell volume, platelet count, total leukocyte count, leukocyte differential count, prothrombine time, fibrinogen concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study (including the satellite groups)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: alkaline aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, creatinine, fasting glucose, phosphorus, total cholesterol, total protein, albumin, chloride, potassium, sodium

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to administration, at monthly intervals and in the last week of dosing and in the last week of the recovery period.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (auditory, visual and proprioceptive stimuli) / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: cranial, thoracic and abdominal cavities were opened and examined macroscopically
HISTOPATHOLOGY: adrenals, aorta, brain (3 sections), epididymides, eye, femur, heart, kidney, liver, lungs (incl. mainstem bronchi), mesenteric lymph node, muscle incl. sciatic nerve, oesophagus, ovaries, pancreas, pituitary, prostate, seminal vesicle, skin incl. mammary glands, small and large intestine (including peyer´s patches), spinal chord (3 levels), spleen, sternum with bone marrow, stomach, submandibular lymph node, testes, thymus, thyroid (incl. parathyroids), trachea, urinary bladder, uterus
Other examinations:
Organ weights of adrenals, brain, epididymides, testes, heart, kidneys, liver, ovaries, spleen, testes and thymus
Statistics:
Body weights, food consumption: Welch t-test

Haematology, coagulation, clinical biochemistry and absolute and relative organ weights: Dunnett´s test

Differential leukocyte count: Mean, range and standard deviation

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
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:
effects observed, treatment-related
Description (incidence and severity):
high dose group (female): fibrinogen concentration of plasma and creatinine content increased, high dose group (male/female): alkaline phosphatase increased, middle and high dose groups (male/female): sreum urea nitrogen increased, all non-adverse
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased kidney weights for high dose males and females, non-adverse
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
intracellular fat and low-grade fatty degenerations of hepatocytes in all male animals, female control, middle dose and high dose groups, non-adverse
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Two animals died shortly after administration due to incorrect gavage shown by lungs filled with blood. None of the animals showed any alterations of their general state of well-being and behaviour at any observation period (few observations were made substance independent and for a short period of time).

BODY WEIGHT AND WEIGHT GAIN
Not affected by the test compound.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Not dose-dependently influenced.

OPHTHALMOSCOPIC EXAMINATION
No alterations.

HAEMATOLOGY
Not influenced.

CLINICAL CHEMISTRY
The fibrinogen concentration of the plasma was increased in the female animals of the high dose group, this was no longer apparent at the end of the treatment-free period.
The activity of alkaline phosphatase of the serum significantly increased in the high dose group, males and femals. This indicates damage to liver cells and/or an increased function rate. This finding was no longer apparent at the end of the treatment-free period.
The serum urea nitrogen was significantly increased in the middle and high dose group of the males and in the high dose group of female animals. The creatinine content was significantly increased in all male and in the high dose group of the female animals. The phosphorus content was significantly increased dose-dependently in all female animals and the sodium content was dose-dependently decreased in the male animals, significantly in the animals of the middle and high dose groups. These effects were no longer apparent at the end of the treatment-free period.

NEUROBEHAVIOUR
No changes in grip strength, motor activity and sensory response.

ORGAN WEIGHTS
Absolute and relative kidney weights were increased in all male animals in the high dose group which was still present after the recovery period. Absolute and relative liver weights were increased in both sexes but this was no longer apparent after the recovery period in females. Other significant differences seem to be incidental.

GROSS PATHOLOGY
No substance-dependent changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Intracellular lipid droplets in hepatocytes of the female animals in the high and mid dose group (5-90% of the observed area) with cell lesions were clearly caused dose-dependently by the test article. There was no special localization of the changes of hepatocytes in the liver lobules. In most cases only low grade intracellular lipopexia occurred in the male animals.
Stomach: Oesophagal part and cardia with multilayered squamous epithelium, leukocyte infiltration in the submucosa and fibrous repair, fibrotic regions in the submucosa of the glandular stomach
Lungs: Atelectactic and emphysemic areas
Thymus: Partial substitution of the parenchyma by fibrinogenesis
Skin: Benign fibrous proliferation
Sciatic nerve: Thickening of the perineurium and thickening of the adventitia of the vessels


Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed up to and including the highest tested dose level

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion