Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
reproductive toxicity, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2004-07-19 to 2004-11-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
1995
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): AE 425/03
- Physical state: weak yellowish, solid
- Lot/batch No.: 11/2003
- Expiration date of the lot/batch: December 2005
- Storage condition of test material: dry, cool and dark

Test animals

Species:
rat
Strain:
other: Crl:CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: (start of adaptation) males 28 days; females 29 days
- Weight at study initiation: males: (start of adaptation) 78.3 - 90.1 g; females: 81.1 - 89.1 g
- Housing: individually in Makrolon cages typ III, (39 x 23 x 15 cm) granulated texture wood beeding
- Diet: ad libitum ssniff R/M-H V 1530; Ssniff Spezialdiäten GmbH, D-59494 Soest
- Water: tap water ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 15
- Air changes (per hr): no data
- Photoperiod: 12 hour dark/light cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: hydroxypropylmethylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate dose levels and was administered
orally by gavage, at a constant volume of
10 mL/kg bw/day for 28 days.
The amount of the test item was adjusted to each animal’s actual body weight daily.
The test item-vehicle mixture was freshly prepared every day.
VEHICLE
- Justification for use and choice of vehicle (if other than water): not given, however 0.8 aqueous hy
droxypropylmethylcellulose is a common vehicle used in toxicity studies not expected to have any ad
verse effects
- Concentration in vehicle: 0.8 aqueous hydroxypropylmethylcellulose
- Amount of vehicle (if gavage): 10 mL/kg bw/ day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To determine the concentration of the test item in the solution, samples of the test item-vehicle mi xtures were taken by the laboratory at study initiation and termination and stored at – 20°C or colder until dispatch for analysis.
Duration of treatment / exposure:
28 days of exposure and a 2-week recovery period
Frequency of treatment:
once daily (7 days per week) for 28 days
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:
5 males and 5 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for this study were selected in agreement with the sponsor based on the 7 day dose-range-finding study in rats.
- Rationale for animal assignment (if not random): a computerised randomisation program was used
- Rationale for selecting satellite groups: not given
- Post-exposure recovery period in satellite groups: 2 – week recovery period; 5 males and 5 females per group for control and high-dose group.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
Observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behavior patterns.
The onset, intensity and duration of any signs were recorded. Additionally, once before the first exposure and once weekly thereafter (1, 2, 3, 8 and 24 hours after administration), detailed clinical observations were made in all animals; in test week 4 these observations were performed prior to any
laboratory investigations. These observations were made outside the home cage in a standard arena
and at the same time, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and
autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking
backwards) were also recorded.
BODY WEIGHT: Yes
- Time schedule for examinations: at the time of allocation of animals to groups, on the day of commencement of treatment and once a week therafter always on the same day of the week.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption per individual rat was recorded on a weekly basis.
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
WATER CONSUMPTION AND COMPOUND INTAKE : yes
Drinking water consumption was monitored by daily visual appraisal throughout the study
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of administration and on test day 28 and at the end
of the recovery period (test day 40)
- Dose groups that were examined: all
The eyes of all animals were examined with a HEINE ophthalmoscope. Prior to examination mydriasis was produced after instillation of MYDRIUM eye drops into conjunctival sacs. The following ocular structures were examined:
Adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, fundus
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at test day 29 all main study animals (5 animals/sex/group); at
day 43 all animals of the recovery period
- Anaesthetic used for blood collection: Yes (identity) ether
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: Haemoglobin content (HGB), erythrocytes (RBC), leucocytes (WBC), differential blood count, reticulocytes, platelets, haematocrit value, thromboplastine time, activated partial thromboplastine time, mean corpuscular volume (MCV), mean corpuscular haemoglobin( MCH), mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at test day 29 all main study animals (5 animals/sex/group); at
day 43 all animals of the recovery period
- Animals fasted: Yes overnight
- How many animals: all
- Parameters examined: Albumin, globulin, A/G ratio, cholesterol total, creatinine, glucose, protein total, urea in blood, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase,
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In test week 4 approximately 1 to 2 hours after dosing and before any blood sampling
- 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), as well as the assessment of grip strength and motor activity assessment was conducted in all animals.

GROSS PATHOLOGY: Yes
All animals were sacrificed under ether anesthesia by cutting the aorta abdominalis, exsanguinated,weighed, dissected and inspected macroscopically under the direction of a pathologist.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 under suitable illumination. The liver and the kidneys were 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 (2), heart, ovary (2), thymus, brain, kidney (2), spleen, epididymis (2), liver, testicle Adrenals, gonads and kidneys were weighted individually and identified as left and right.
HISTOPATHOLOGY: Yes
Blood smears were prepared for examination of pathological changes from all animals but were examined and evaluated only depending on necropsy findings.
The following organs of parts of organs of all animals were fixed in 7 % buffered formalin. The eyes were preserved in Davidson´s solutions for optimum fixation:
Adrenal (2), aorta abdominalis, bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), epididymis (2), 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), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles), lymph node (1cervical), lymph node (1 mesenteric), mammary gland, nerve sciatic, oesophagus, ovary (2), pancreas, pituitary, prostate, salivary glands (mandibular, parotid, sublingual), skin (left flank), spinal cord (3 sections), spleen, stomach, testicle (2), thymus, thyroid (2) incl. parthyroids), tissue masses or
tumors (including regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts), vagina
The afore-listed organs of all animals of control and high dose groups 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 they were in the plane of section and in all cases where they were noted as grossly enlarged.
Statistics:
The following statistical methods were used:
Student's t-test for numerical functional tests (p ≤ 0.01)
Multiple t-test based on DUNNETT, C. W. for body weight, food consumption, haematology, clinical biochemistry, relative organ weights (p ≤ 0.01)
Exact test of R. A. Fischer for histopathology (p ≤ 0.05)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
None of the rats treated with 100, 300 or 1000 mg/kg bw /day showed any clinical signs of systemic toxicity. The faeces of all animals were of normal consistency throughout the experimental period.
Mortality:
no mortality observed
Description (incidence):
No mortality was noted at any of the tested dose levels 100, 300 or 1000 mg/kg bw/day during the treatment or recovery period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight of the male and female rats treated with 100, 300 or 1000 mg/kg bw/day test substance was not influenced as compared to the control animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the relative food intake of the male and female rats treated with either 100, 300 or 1000 mg/kg bw/day test substance as compared to the control animals.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The visual appraisal of the drinking water consumption revealed not differences between the control and the test item – treated animals.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No pathological changes were noted on the adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus as revealed by ophthalmological observation prior to the start of administration, at the end of test week 4 (on test day 29) and at the end of the recovery period (on test day 40).
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was observed for any of the haematological parameters examined in the animals treated with 100, 300 or 1000 mg/kg bw/day.
No test item-related influence was observed for the haemoglobin content, the number of erythrocytes, leucocytes, reticulocytes and platelets, the haematocrit value, the thromboplastin time (TPT) and the activated partial thromboplastin time (aPTT), the mean corpuscular haemoglobin concentration (MCHC). Evaluation of the differential blood count revealed no remarkable test item-related deviations.Statistically significant differences (at p ≤ 0.01) in haematological parameter which are not considered
to be test item-related:
A decrease in TPT and MCH value at test day 29 in the female 1000 mg/kg bw/day test group was considered to be a slight alteration in comparison to control animals without biological relevance.
A decrease in MCV value at test day 29 in the female 100 and 1000 mg/kg bw/day test groups was considered to be a slight alteration in comparison to control animals without biological relevance andlacking dose dependency.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related influence was observed for any of the biochemical parameters examined in the animals treated with 100, 300 and 1000 mg/kg bw/day test substance.
No test item related influence was noted on the plasma levels of albumin, globulin, cholesterol, creatinine, glucose, protein, urea in blood, potassium, sodium and the albumin/globulin ratio. The activities of alanine aminotransferase (ALAT), alkaline phosphatase (aP) and aspartate aminotransferase (ASAT) were within normal limits.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation in test week 4 revealed no changes in any of the parameters examined for the rats treated with 100, 300 or 1000 mg/kg bw/day test substance. No test item-related effects on the fore- and hindlimb grip strength and the spontaneous motility was noted for the male and female an
imals of all groups. Statistically significant differences (at p ≤ 0.01) in functional observations which are not considered to be test item-related: An increase in body temperature at test week 4 in the female 100 mg/kg bw/day test group was observed with lacking dose dependency.
Increased forelimb grip strength was observed at test week 4 in all male test groups, the slight alteration in comparison to control animals was considered to be without biological relevance.Increased hindlimb grip strength was observed at test week 4 in male 300 and 1000 mg/kg bw/day test groups and female 300 mg/kg bw/day test group. The slight alteration in comparison to control animals was considered to be without biological relevance and for the females with lacking dose dependence.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Treatment and recovery period:
All microscopic findings noted were within the normal range for animals of this strain and age and did not distinguish distinctly treated rats from control rats. Type, incidence and severity of these findings noted were not increased in the test item-treated animals compared to the controls.
The test item at a dose level of 1000 mg/kg bw/day for a treatment period of 4 weeks did not produce any histopathological evidence of any systemic toxic effect in either sex.
Histopathological findings: neoplastic:
not examined

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: other: highest tested dose, no treatment related effects at all

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

Effect levels (F1)

Remarks on result:
not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Analytical dose verification:

The analytical verification of dosing solutions demonstrated agreement of actual initial concentrations with nominal test concentrations, and the dosing suspensions were documented to be sufficiently stable and homogenous over the entire dosing period.

Applicant's summary and conclusion