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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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Germany. Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: 230.4 - 260.2 g (males), 138.8 - 201.3 g (females)
- Housing: in groups of two, three or four in Makrolon type-4 cages with wire mesh tops
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C (batch no. 39/12) rat/mouse maintenance diet (Provimi Kliba AG, Switzerland), ad libitum
- Water (e.g. ad libitum): community tap water from Itingen, ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES:
From: 14.11.2012 To: 21.03.2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.1% in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 4, 15 or 45 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): 120M0216V
- Purity: 0.1% in water

The dose formulations were prepared wekly. Homogenity of the test item 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:
On the first treatment day and once during weeks 5 and 12, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of homogeintiy and concentration. Duplicate samples of about 1 g of each concentration were taken four hours after commencement of dosing to confirm stability.
The analysis was performed by the responsible using a HPLC method.
Duration of treatment / exposure:
main study:
91/92 days
recovery period:
29 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
450 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
main groups: 10
recovery groups: 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a previous 28 d toxicity study in rats
- Post-exposure recovery period in satellite groups:recovery period 29 days; 5 animals/sex/dose; control and high dose group
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
twice daily on days 1 to 3; once daliy thereafter

DETAILED CLINICAL OBSERVATIONS: Yes
once weekly during weeks 1 to 3, not performed during recovery

BODY WEIGHT: Yes
once weekly

FOOD CONSUMPTION:
weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
once during the acclimatization period; once during week 13 in animals of the control and the high dose group, as well as in animals of the middle dose group if test item related changes are seen in the high dose, using a direct ophthalmoscope

HAEMATOLOGY: Yes
after week 13 and week 17; blood samples were drawn from the retro-orbital plexus from al animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approx. 18 h before blood but allowed access to water ad libitum.
The following hematology parameters were determined: erythrocyte count, hemoglobin, hemarocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, reticulcyte count, reticulocyte maturity index (low, medium, high fluorescence), leukocyte count (total), neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells, platelet count, prothrmbin time, activated partioal thromboplastin time.

CLINICAL CHEMISTRY: Yes
Collection details see above. The following clinical biochemistry parameters were determined: glucose, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, aspartate amintransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, bile acids, gamma-glutamyl-tersnferase, creatine kinase, sodium, potassium, chloride, calcium, phophorus, protein (total), albumin, globulin, albumin/globulin ratio

URINALYSIS: Yes
after week 13 and 17; urine was collected during 18 h fasting period. The following urine parameters were determined: urine volume, specific gravity, color, appearance, pH value, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, erythrocytes, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
During weeks 13 and 17;
Grip strength: Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge. The animals were placed with the forepaws inside a triangular grasping ring and with the hindpaws 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.
Locomotor activity: Locomotor activity was measured quantitatvely with AMS Föhr Medical Instruments and DeMe Tec GmbH Activity Monitor System. Animals were monitored during treatment weeks 13 and 17 for a 60minute period and the total activity of this time period was recorded.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
main groups: after 13 weeks
recovery groups: after 17 weeks

HISTOPATHOLOGY: Yes
The following organs/tissues were examined: adrenal glands, aorta, bone marrow (femur), cecum, colon, duodenum, epididymides, esophagus, eyes/optic nerve, heart including auricles, ileum with Peyer's patches, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lungs, lymph nodes (mesenteric and mandibular), mammary gland area, ovaries, pancreas, pituitary gland, prostate gland and seminal vesicles incl. coagulating glands, rectum, salivary glands (mandibulr and sublingual), sciatic nerve, skin, spinal cord (cervical, midthoracic and lumbar), spleen, stomach, testes, thymus, thyroid (incl. parathyroid gland), trachea, urinary bladder, uterus incl. oviducts, cervix and vagina, all gross lesions.

Organ weights:
Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus
Other examinations:
none
Statistics:
The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, locomotor activity, grip strength, ophthalmoscopic examinations, organ weights and ratios as well as macroscpic findings:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if variables could be assumed to follow a normal distribution for the comparison 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 when the data could not be assumed to follow a normal distribution.
- Fisher's exact test

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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
- Mortality/Viability:
One male treated with 150 mg/kg bw/d was found dead on treatment day 60, but was considered to an incidental death without toxicological relevance. All other animals survived until the scheduled necropsy.

- Clinical signs:
No cllinical signs were noted in males and females treated with 50 mg/kg bw/d of the test item during the course of the study. No test item relatated signs were noted in males and females treated with 150 or 450 mg/kg bw/d of the test item during the treatment and the recovery period.

- Functional observational battery:
No test item related clinical signs were noted in males and females of all treated groups during the treatment and recovery periods.

- Grip strength:
No test item tleated changes of the mean fore- and hindlimb grip strength were noted in males and females when compared with controls during the treatment and recovery periods.

- Locomotor activity:
No test item related differences of mean locomotor activity were noted when comparing controls and test item treated animals in treatment week 13 and in the recovery period.

- Food consumption:
No test item related differences in mean absolute and relative food consumption were noted in test item treated males and females when compared with controls during the treatment and recovery period.

- Body weights:
No test item related changes of the mean body weights and the mean body weight gain of test item treated males and females were noted when compared with controls during the treatment and recovery period.

- Clinical laboratory investigations:
No test item related differences in parameters of hematology, clinical biochemistry and urinalysis were noted in test item treated males and females when compared with control animals at the end of the treatment period and at the end of the treatment free recovery period.

- Organ weights:
No test item related differences in the mean organ weights, mean organ to body weight ratios and mean organ to brain weight ratios were noted in males and females of all test item treated groups when compared with controls at the end of the treatment and at the end of the recovery period.

- Macroscopic/microscopic findings:
No test item related macroscopic findings were noted in males and females. Ther were no treatment-related microscopic findings. All microscopic findings recorded in this study were considered to be within the normal range of background alterations that is seen in untreated animals of this age and strain.












Effect levels

Dose descriptor:
NOEL
Effect level:
450 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the observations made in this 90 d oral toxicity study in rats the NOEL was considered to be 450 mg/kg bw/d (the highest dose tested).
Executive summary:

Oral administration of the test item to Sprague Dawley rats at doses of 50, 150 and 450 mg/kg bw/ d, for 91/92 days resulted in no deaths of toxicological relevance, no test item-related findings during daily observations or weekly observations (weeks 1 -12), during the functional observation battery (weeks 13 and 17), no differences of toxicological relevance in the fore- and hindlimb grip strength values or locomotor activity, no differences of toxicological relevance in themean food consumption or mean body weight development, no test item-related differences in the ophthalmoscopy, minor test item-unrelated differences in hematology, clinical biochemistry and urinalysis, no toxicologically relevant differences in mean absolute and relative organ weights, and no macroscopical findings of toxicological relevance.

Based on the the results of this study, 450 mg/kg bw/d of the test item was established as NOEL.