Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-03-10 to 2006-11-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD test guideline 408

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21 August 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sulzfeld/Germany
- Age at study initiation: 36-37 days (incl. 9 days acclimation period)
- Weight at study initiation: Males: 72.9 - 84.9 g
Females: 70.2 - 80.9 g
- Fasting period before study: no
- Housing: MAKROLON cages (type III) with a basal surface of approx. 39 x 23 cm and a height of approx. 15 cm
- Diet: ssniff® R/M-H V1530 ad libitum
- Water: ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3
- Humidity: 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: soybean oil
Details on oral exposure:
VEHICLE
- Amount of vehicle: 5 mL/kg bw/day (administration voulme)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each test or reference item that was mixed with a vehicle, tests by appropriate analytical methods were conducted to determine the concentration, stability and homogeneity of the test or reference item in the solution. For the analysis of the test item-vehicle mixtures, samples of approximately 10 mL were taken at the following times and stored at -20°C or colder until analysis.

At study initiation:
- Analysis of stability and concentration
Immediately after preparation of the mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature (3 samples/dose level group). Total number of samples: 9
- Homogeneity
At the start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples/dose level group). Total number of samples: 9

At study termination:
- Analysis of concentration
During treatment with the test item always before administration to the last animal/dose level group (1 sample/dose level group)
Total number of samples: 3

The samples were labelled with study number, species, type of sample, concentration, test day, sampling time and date.
The samples were analysed according to a spectrophotometer method revalidated by LPT.
The following parameters were determined during re-validation:
- linearity
- accuracy
- precision
- sensitivity
- specificity
The results of the validation revealed that the method used was reproducible and led to reliable results.
The analysis of test substance in the test item-vehicle mixtures confirmed that the formulations used for the administration in groups 2 to 4 were correctly prepared and stable for at least 24 hours after storage at room temperature. The results of 95.4% to 106.1% of the nominal value were well within the admissible limits of 90% to 110%.
Duration of treatment / exposure:
90 d
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on available toxicological data.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Parameters examined: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before adminsitration and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at start and termination
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At study termination
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, reticulocytes, platelets, differential blood count (rel. and abs.), haematocrit value, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemogoblin (concentration),

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At study termination
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: albumin, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), triglycerides, uric acid, urea, calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: At study termination
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes (overnight)
- Parameters examined: pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin, nitrite, microscopic examinations, colour and turbidity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in week 13
- Dose groups that were examined: all
- Battery of functions tested: screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli); grip strength; motor activity

FOOD and DRINKING WATER CONSUMPTION
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week. Drinking water consumption was monitored daily by visual appraisal throughout the study.

OESTRUS CYCLE
At the end of the study (weeks 12 and 13), vaginal lavages were taken daily from all female animals for periods of 2 weeks each. The stages of the oestrus cycles observed in each vaginal lavage were recorded.

SPERM COUNT, VIABILITY, MOTILITY:
From one testicle and epididymis a sperm count was carried out, the sperm viability was determined and the sperm morphology was examined for the control and treated animals.

Blood sampling for determination of plasma levels (at termination in all animals under light ether anaesthesia)
Sacrifice and pathology:
SACRIFICE:
Starting on test day 91 (one day after the last administration) the animals were dissected following a randomisation scheme. Animals not dissected on test day 91 were dosed again until one day before sacrifice. The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis,
exsanguinated, weighed, dissected and inspected macroscopically under the direction of a pathologist.

PATHOLOGY:
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weights of the following organs of all animals were determined before fixation:
adrenal gland (2), heart, ovary (2), thymus,brain, kidney (2), spleen, uterus, epididymis (1), liver, testicle (1)

The following organs or parts of organs of all animals (including deceased or sacrificedanimals) were fixed in 7% buffered formalin. The eyes were preserved in Davidson's solution.

adrenal (2)
aorta abdominalis
bone marrow (os femoris)
brain (3 levels: cerebrum, cerebellum, medulla/pons)
epididymis (1)
eye with optic nerve (2)
gross lesions observed
heart (3 levels: right and left ventricle, septum)
intestine, large (colon, rectum)
intestine, small (duodenum, jejunum,
ileum, incl. Peyer's patches, Swiss roll method)
kidney and ureter (2)
liver
lungs (with mainstem bronchi and bronchioles [preserved by inflation with fixative and then immersion])
lymph node (cervical) (1)
lymph node (mesenteric) (1)
mammary gland
nerve (sciatic)
oesophagus
ovary (2)
pancreas
pituitary
prostate
salivary glands (mandibular, parotid and sublingual gland)
skin (left flank)
spinal cord (3 levels: cervical, mid-thoracic, lumbar)
spleen
stomach
testicle (1)
thymus
thyroid (2) (incl. parathyroids)
tissue masses or tumours (including regional lymph nodes)
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix and oviducts)
vagina
The afore-listed organs of all animals of groups 1 and 4 were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were made and stained with scarlet R.
Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plan of section and in all cases where they were noted as grossly enlarged.

Spermiogram:
From one testicle and epididymis a sperm count was carried out, the sperm viability was determined and the sperm morphology was examined for the control and treated animals according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Statistics:
Student's t-test
ANOVA with Dunnett's post-hoc test
Fisher's exact test

Results and discussion

Results of examinations

Details on results:
MORTALITY
No test item-related mortality occurred during the study. However, female animals no. 31 and 40 (100 mg), 60 (300 mg ) and 80
(1000 mg) died during laboratory examinations on the last test day after blood withdrawal due to the ether narcosis. This is regarded to be spontaneous and the animals were handled as terminal sacrificed animals.

URINALYSIS
The male and female animals treated with 300 or 1000 mg/kg bw/day revealed a dose dependent decrease of urinary pH value by up to 11 % (statistically significant at p ≤ 0.01 for males of both dose levels and for the female at the high dose). This effect is considered to be potentially caused by the low pH of the registered substance or by a metabolite eliminated at large doses via the urine.

ORGAN WEIGHTS
Treatment with 1000 mg/kg bw/day caused an increase in the absolute and relative liver weights (both sexes). The increase is considered to be a non-specific adaptive change to the high work load of the liver caused by a dose level of 1000 mg/kg bw/day.

OESTRUS CYLE:
No test item-related changes in the oestrus cycle were found in the evaluated females at any dose during the observation periods (test week 12 + 13).

SPERM COUNT, VIABILITY, MOTILITY:
The number of ultrasound-resistant spermatids per gram of testicular tissue was not influenced by any dose.
No test item-related changes were noted in the numbers of motile spermatozoa in the cauda epididymis for the male animals treated with 100, 300 or 1000 mg test substance/kg bw/day. The male animals treated with 100, 300 or 1000 mg/kg bw/day showed 99.9 to 100 % morphologically normal spermatids.

Effect levels

Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: all observations

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion