Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-08-27 to 2008-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2008

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:
2001/56/EEC
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: CD/Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: 26 days; females : 27 days
- Weight at study initiation: males: 72.6 - 85.0 g; females: 70.1 - 79.2 g
- Fasting period before study: no
- Housing: animals were kept singly in MAKROLON cages (type III) with a basal surface of approx. 39 x 23 cm and a height of approx. 15 cm
- Diet: Commercial ssniff® R/M-H V1530 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany), ad libitum
- Water: Commercially available drinking water according to "Deutsche Trinkwasserverordnung 2001", ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 55 +/- 15 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2007-09-13 To: 2007-12-12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item-vehicle mixtures were freshly prepared every day. The test item was dissolved in the vehicle to the appropriate concentrations and was administered orally by gavage at a constant volume of 5 mL/kg b.w. daily for 90 days. The control animals received the vehicle for 90 days in the same manner. The amount of the test item was adjusted to the animal's current body weight daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In an analytical study, the concentrations of the test item were determined in aqueous formulation solutions obtained from a preclinical study. The aqueous formulation samples were analyzed using a validated high performance liquid chromatography assay with mass spectrometric detection (LC-MS). The in-study validation was performed with respect to the validation parameters accuracy, precision and selectivity, using data from blank matrix, calibration standards and quality control (QC) samples.
Calibrated Range [mg/L]: 0.404 to 20.195
Defined lower limit of quantification (LLOQ) [mg/L]: 0.404
Precision (%) for QC samples: below 13
Accuracy (%) for QC samples: better than -7
It was concluded that the overall performance of the method was good and that, therefore, the results obtained for the samples were of the required integrity and quality.
Duration of treatment / exposure:
90 d
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males/10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Route of administration: Oral, by gavage
- Frequency of administration: Once daily for 90 days
- Vehicle: Tap water
- Administration volume: 5 mL/kg b.w./day
- Selection of route of administration: According to OECD guideline 408 and EC guideline B.26.
The test item-vehicle mixtures were freshly prepared every day. The test item was dissolved in the vehicle to the appropriate concentrations and was administered orally by gavage at a constant volume of 5 mL/kg b.w. daily for 90 days. The amount of the test item was adjusted to the animal's current body weight daily.
Positive control:
The control animals received the vehicle for 90 days in the same manner.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: signs of behavioural changes, reaction to treatment or illness.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before first exposure, one week thereafter, test week 13.

BODY WEIGHT: Yes
- Time schedule for examinations: on day of allocation, on day of commencen,ment of the study and weekly thereafter.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of administration and at study termination in test week 13:
- Dose groups that were examined: all animals

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 animals
- Parameters checked: haemoglobin content, erythrocytes, leucocytes, reticulocytes, platelets, differential blood count relative and absolute (Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes, monocytes and large unstained cells), haematocrit value, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration;

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes
- How many animals: all
- Parameters checked: albumin, globulin, albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), triglycerides, urea
(blood urea nitrogen, BUN), calcium, chloride, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH);

URINALYSIS: Yes
- Time schedule for collection of urine: at study termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (Hb), nitrite, sediment;

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: test week 13
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other: righting reflex, body temperature, salivation, startle reflex, respiration, mouth-breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function;

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following organs or parts of organs of all animals (including deceased or sacrificed animals) were fixed in 7% buffered formalin. The eyes were preserved in Davidson's solution.
Adrenal gland (2), aorta abdominalis, bone marrow (os femoris) brain (3 levels: cerebrum, cerebellum, medulla/pons), coagulating gland with seminal vesicle, 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, 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 (male and female), 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 (2), 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.
Statistics:
The test item-treated groups (2 - 4) were compared with the control group (1).
The following statistical methods were used:
- STUDENT's t-test:
All numerical functional tests / urinalysis (p ≤ 0.01). The following limit was used: p ≤ 0.01 ≙ t = 2.878 for 18 degrees of freedom.
- Multiple t-test based on DUNNETT, C. W. New tables for multiple Comparisons with a control. Biometrics 482-491 (Sep 1964):
Body weight / food consumption/ haematology / clinical biochemistry / organ weights (p ≤ 0.01) The following limit was used: p ≤ 0.01 ≙ t = 3.09 for 36 degrees of freedom.
- Exact test of R.A. FISHER: Histopathology (p ≤ 0.05)
These statistical procedures were used for all data. Significantly different data are indicated.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No test item-related effect on behaviour and external appearance was noted for the male and female rats treated orally with 100, 300 or 1000 mg/kg bw/day of the test item. No mortality occurred during the course of the study.

BODY WEIGHT AND WEIGHT GAIN: Body weights were not influenced in male and female rats treated with 100, 300 or 1000 mg/kg b.w./day of the test item. Body weight gain and body weight at autopsy were similar in all groups.

FOOD AND DRINKING WATER CONSUMPTION: No test item-related influence was observed on the food consumption of male and female rats treated with either 100, 300 or 1000 mg/kg b.w./day of the test item. The visual appraisal of the drinking water consumption revealed no differences between the control and the test item-treated animals.

OPHTHALMOSCOPIC EXAMINATION: Ophthalmological examination of the ocular structures revealed no lesions of the eyes or the optic region in the rats at the end of the 90-day treatment period with either 100, 300 or 1000 mg/kg b.w./day of the test item.

HAEMATOLOGY: No test item-related changes were noted for the haematological parameters of male and female animals treated with 100, 300 or 1000 mg/kg b.w./day of the test item.

CLINICAL CHEMISTRY: No test item-related changes were noted on the biochemical parameters of male and female animals treated with 100 or 300 mg/kg b.w./day or of the female animals at 1000 mg/kg b.w./day of the test item. The following test item-related changes were noted for the male rats at 1000 mg/kg b.w./day test item compared to the control: males: bilirubin, total: - 21 %. This is regarded as an isolated finding without any correlation to other haematological parameters.

URINALYSIS: Treatment with 100, 300 or 1000 mg/kg b.w./day of the test item did not lead to any test item-related changes of the urinary status compared with the control group at the end of the treatment period.

NEUROBEHAVIOUR: The parameters of functional observations did not reveal any test item-related influence for the rats treated with 100, 300 or 1000 mg/kg b.w./day of the test item. No influences in fore- and hindlimb strength or spontaneous motility were noted in any animals treated with the test item.

ORGAN WEIGHTS: No test item-related changes were noted for the relative and absolute organ weights of male and female animals treated with 100 or 300 mg/kg b.w. or for the male rats at 1000 mg/kg b.w./day of the test item. Slightly increased relative and absolute uterus weights (by 23%, statistically not significant) were noted for the high dosed female animals treated with 1000 mg/kg b.w of the test item. These findings are considered to be test item-related.

GROSS PATHOLOGY: At necropsy, no test item-related changes were noted in the rats treated with 100, 300 or 1000 mg/kg b.w./day of the test item.

HISTOPATHOLOGY: NON-NEOPLASTIC: A minimal to moderate degree (10/10 male animals) and a minimal to mild degree (10/10 female animals) of swollen gastric chief cells were noted in the pars glandularis of the stomach of the animals treated with 1000 mg/kg b.w./day of the test item. However, a minimal to mild degree of swollen gastric chief cells was also noted in 8/10 male animals and 7/10 female animals of the control. The severity of this finding was distinctly increased for the male animals treated with 1000 mg/kg b.w./day of the test item compared to the control group. Therefore, this finding is regarded to be test item-related. All further findings recorded in this study are commonly encountered in rats of this strain and age. Type, incidence, and severity of the lesions recorded were not increased in the treated animals as compared to the control animals.

OTHER FINDINGS: None.

Effect levels

open allclose all
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: systemic changes
Dose descriptor:
NOEL
Effect level:
> 300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: systemic changes

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, the NOEL was 300 mg/kg bw and the NOAEL was 1000 mg/kg bw for both sexes.
Executive summary:

The test item was investigated in accordance with OECD 408 under GLP conditions. 10 male or female CD/Crl (SD) rats received vehicle (water), 100, 300, or 1000 mg test item/kg bw for 90 consecutive days. Body weight, food consumption, functional observational battery, organ weights, clinical chemistry, hematology, urinalysis, macroscopical findings, histopathology were recorded. Isolated but statistically significant systemic changes at 300 mg/kg bw in males and females were either not dose dependant or were not accompanied by corresponding effects (clinical chemistry, histopathology), These effects were not considered to be toxicologically relevant. Therefore, the NOAEL was determined to be 1000 mg/kg bw.