Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 12, 2006 - Jun 22, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:

Purity 99.9%

Test animals

Species:
rat
Strain:
other: HanRcc:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Age at delivery 6 weeks
Body weight range at acclimatization
Males: 135.7 – 163.1 grams (mean 147.8 grams)
Females: 114.5 – 130.8 grams (mean 121.5 grams)

Acclimatization
Under test conditions after health examination. Only animals without any visible signs of illness were used forthe study.

HUSBANDRYCONDITIONS
Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environmental conditions (temperature range: 22 ± 3 °C; relative humidity range: 30-70 %). Values outside of these ranges occasionally occurred, usually following roomcleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

ACCOMODATION
In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).

DIET:
Pelleted standard Provimi Kliba 3433 (batch nos 48/06, 567/06) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.

WATER:
Community tap-water from Itingen was available ad libitum in water bottles. None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
DOSE FORMULATION
Frequency of dose formulation weekly, based upon stability analysis results of RCC
Study No. B05501
The test item was weighed into a tared glass beaker on a suitable precision balance and the
vehicle, PEG 300, added (weight:volume). The mixtures were prepared using a magnetic stirrer.
Homogeneity of the test item in the vehicle was maintained during the daily administration
period using a magnetic stirrer.
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. Analyses were performed by the Principal Investigator of the analytical phase (see Responsibilities, page 6), according to a HPLC analytical method supplied by the Sponsor and adapted during RCC Study Number B05501. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The dose formulations were delivered under ambient conditions. Samples of dose formulations were not discarded without the written authorization of the study director.

The identity of the test material was confirmed by its retention time which was similar to that measured in the working standards. The test item content in all samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution of the test material in PEG 300 was demonstrated. The application formulations were considered to be stable for at least 2 hours and 7 days when kept under storage conditions.

In conclusion, the results obtained within this phase confirm the correct preparation and storage of application formulations during the conduct of this study.
Duration of treatment / exposure:
Method Oral, by gavage.
Rationale Administration by gavage is a common and accepted route of exposure for studies of this type.
Daily dose levels
Group 1: 0 mg/kg body weight
Group 2: 40 mg/kg body weight
Group 3: 200 mg/kg body weight
Group 4: 1000 mg/kg body weight

Rationale for dose level selection Based upon results of a 5-Day Dose Range Finding Study (RCC No. B05501) and the LD50 oral for rats provided by
the sponsor (>2000 mg/kg)

Frequency of administration Once daily
Dose volume 5 ml/kg body weight
Duration of acclimatization period 7 days
Duration of treatment 28 days
Duration of recovery 14 days
Frequency of treatment:
daily for 28 days
Doses / concentrations
Remarks:
Doses / Concentrations:0, 40, 200 1000 mg/kg bwBasis:actual ingested
No. of animals per sex per dose:
Allocation
And
Dose Levels Group 1: 0 mg/kg/day Group 2 40 mg/kg/day Group 3 200 mg/kg/day Group 4 1000 mg/kg/day
Males A 1 - 5 11 - 15 16 - 20 21 – 25
Males B 6 - 10 26 - 30

Females A 31 – 35 41 - 45 46 - 50 51 – 55
Females B 36 - 40 56 - 60
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
MORTALITY / VIABILITY
Observations for mortality/viability were recorded twice daily.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
The animals were observed for clinical signs once before commencement of administration;
twice daily on days 1-3; once daily on days 4-28, and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
The animals were observed in their home cages, outside their home cages in a standard arena
and in the hand. These observations were performed once before commencement of
administration and once weekly in weeks 1-3 thereafter.

FOOD CONSUMPTION
The food consumption was recorded once during the pretest period and once weekly thereafter,
using an on-line electronic recording system consisting of a Mettler balance connected to the
RCC computer.

BODY WEIGHTS
Body weights were recorded weekly during pretest, treatment and recovery and before
necropsy, using an on-line electronic recording system consisting of a Mettler balance
connected to the RCC computer.

FUNCTIONAL OBSERVATIONAL BATTERY
During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
The results of the Functional Observational Battery are presented in the summary and
individual tables of the Detailed Clinical Observations (Weekly) under week 4. This method of
data presentation permits a clear evaluation and assessment of weekly clinical signs observed
during the study.

GRIP STRENGTH
Forelimb and hind limb grip strength measurements were performed using a push-pull strain
gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular
grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the
animals were held towards the base of the tail and steadily pulled away or towards the ring until
the grip was broken. Each measurement was repeated three times, the means were calculated
and recorded.


LOCOMOTOR ACTIVITY
Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr
Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were
monitored during the fourth treatment week for a 60-minute period and the total activity of this
time period was recorded.
Low beams count was reported in 10-minute intervals as well as the total activity of the
measuring period.

CLINICAL LABORATORY INVESTIGATIONS
Blood and urine sampling:
after 4 weeks 16-JAN-2007 (Allocation A and B)
after 6 weeks 30-JAN-2007 (Allocation B)
Blood samples for hematology and clinical biochemistry were collected from all animals under
light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately
18 hours before blood sampling but allowed access to water ad libitum. Blood samples were
collected early in the working day to reduce biological variation caused by circadian rhythms.
Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary
tube.

Urine was collected during the 18-hour fasting period into a specimen vial.
The assays were performed at RCC Ltd (Füllinsdorf) under internal laboratory quality control
conditions to assure reliable test results.
In the summary and individual tables the names of some parameters have been abbreviated.

Clinical laboratory data are expressed, with a few exceptions, in general accordance with the
International System of Units (SI).



HEMATOLOGY
The following hematology parameters were determined:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Platelet (thrombocyte) count
Reticulocyte count
Reticulocyte maturity index
Methemoglobin
Total leukocyte count
Differential leukocyte count
Coagulation:
Thromboplastin time
Activated partial thromboplastin time

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Creatine kinase
Alkaline phosphatase
Gamma-glutamyl-transferase
Sodium
Potassium
Chloride
Calcium
Phosphorus inorganic
Protein, total
Albumin
Globulin
Albumin/Globulin ratio
Bile acids

URINALYSIS
The following urinalysis parameters were determined:
Volume (18 hours)
Specific gravity (relative density)
Color
Appearance
pH
Nitrite
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Erythrocytes
Leukocytes



Sacrifice and pathology:
PATHOLOGY

NECROPSY
Sacrifice:
after 4 weeks 16-JAN-2007 (Allocation A)
after 6 weeks 30-JAN-2007 (Allocation B)
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were
recorded.
All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed
by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and,
unless otherwise indicated, fixed in neutral phosphate buffered 4% formaldehyde solution.
Additional tissues (such as ear tattoo) were retained in accordance with standard operating
procedures but will not be processed or examined further.

Section A (examined in HISTOPATHOLOGY)

Adrenal glands
Bone marrow (femur)
Brain (at least 3 levels)
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Heart
Ileum, with Peyer's patches
Jejunum with Peyer's patches
Kidneys
Liver
Lungs, filled w/formalin at necropsy
Lymph nodes - mesenteric, mandibular
Ovaries
Prostate gland (incl. coagulating gland)
Rectum
Sciatic nerve
Seminal vesicles
Spinal cord - cervical, midthoracic, lumbar
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland, if possible)
Trachea
Urinary bladder, filled w/formalin at necropsy
Uterus
Vagina
Gross lesions

Section B (not examined in HISTOPATHOLOGY)

Aorta
Bone (sternum, femur including joint)
Esophagus
Eyes w/optic nerve (fixed in Davidson's solution)
Harderian gland (fixed in Davidson's solution)
Larynx
Lacrimal gland, exorbital
Mammary gland area
Nasal cavity
Pancreas
Pituitary gland
Salivary glands - mandibular, sublingual
Skeletal muscle
Skin
Tongue


ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy:
Brain Thymus Spleen Ovaries
Heart Kidneys Testes
Liver Adrenals Epididymides

The organ to terminal body weight ratios as well as organ to brain weight ratios were
determined.
The determination of the terminal body weight was performed immediately prior to necropsy.

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 Necropsy section A were collected at scheduled sacrifice from the animals of control and high-dose groups were
examined by a pathologist.
Because treatment-related morphologic changes were detected in the livers of high-dose
animals, the livers from animals of the mid- and low-dose group were examined.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity,body weight, clinical laboratory data, organ weights, and ratios:• The Dunnett-test (many to one t-test) based on a pooled variance estimate wereapplied if the variables could be assumed to follow a normal distribution for thecomparison of the treated groups and the control groups for each sex.• The Steel-test (many-one rank test) was applied instead of the Dunnett-test whenthe data can not be assumed to follow a normal distribution.• Fisher's exact-test was applied to the macroscopic findings.• Student’s t-test was applied to grip strength and locomotor activity data

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Localized hair loss was seen in two males of the control group during treatment. One of those
two males additionally showed scabs during treatment. One female treated with
1000 mg/kg/day showed hair loss in several locations during treatment. One male treated with
1000 mg/kg/day showed hair loss, scabs and an injured eye during recovery.
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 specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The changes were without statistical significance or dose relation unless
otherwise mentioned. Thus, the often contrary findings were considered not to be test itemrelated.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The changes were without statistical significance or dose relation unless
otherwise mentioned. Thus, the often contrary findings were considered not to be test itemrelated.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 4 weeks of treatment, a slight increase in relative densitiy of urine together with slightly
decreased volume was noted in males treated with 1000 mg/kg/day. These findings were
statistically not significant and showed no dose-relation. Thus, they were considered not to be
test item-related.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The changes noted were without dose relation and / or contrary across sexes and time points.
Thus, they are considered to be incidental.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 4 weeks of treatment, findings such as adherent liver lobes, reduced size of testes and
epididymides, constriction of the spleen, dilation or watery cysts of the uterus, foci in external
lacrimal glands, and alopecia each occurred in single animals of different groups.
After recovery, one male of the control group had foci in the stomach. Foci in the thymus were
noted in one female treated with 1000 mg/kg/day.
These findings were within the range of normal background lesions, which may be commonly
recorded in animals of this strain and age.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Oral application of the test material at the dose levels of 200 and 1000 mg/kg/day
induced minimal hypertrophy of centrilobular hepatocytes in the liver of some males and
females. This liver change was reversible within the treatment-free recovery period. Thus, this
finding was considered to represent an adaptive change and to be non-adverse.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 1000 mg/kg body weight/day of the test material could be established as the no-observed-adverse-effect-level (NOAEL). Based on this study no classification or labelling is warranted.
Executive summary:

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 407.

Based on the results of this study, 1000 mg/kg body weight/day of the test material could be established as the no-observed-adverse-effect-level (NOAEL). Based on this study no classification or labelling is warranted.