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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: according to OECD guideline 408 under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): C-SAT 080029
- Physical state: colourless liquid
- Analytical purity: 99.1%
- Composition of test material, percentage of components: dioctylether: 99.1%
- Purity test date: February 27, 2008
- Lot/batch No.: CE72530027
- Expiration date of the lot/batch: September 09, 2009
- Stability under test conditions: September 09, 2009
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: at start of adaptation: males: 26 days, females: 27 days
- Weight at study initiation: at start of adaptation: males: 72.9-84.0 g; females: 70.9-82.9 g
- Fasting period before study: no
- Housing: singly
- Diet (e.g. ad libitum): conventional laboratory diet ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 9 adaptation days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C (maximum range)
- Humidity (%): 55% +/- 15% (maximum range)
- Air changes (per hr): 12 - 18 times
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: June 20, 2008 To: September 18, 2008 (main study animals) or October 30, 2008 (recovery animals)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item-vehicle mixtures were freshly prepared every day. The test item was suspended in the vehicle (sunflower oil) to the appropriate
concentrations and was administered orally by gavage at a constant volume of 5 mL/kg b.w. daily for 90 days.


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:


VEHICLE
- Justification for use and choice of vehicle (if other than water): most suitable vehicle
- Concentration in vehicle: 0.02, 0.06, 0.2 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg b.w.day
- Lot/batch no. (if required): product no. 88921, LOT: 1379622
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of the concentration of C-SAT 080029 in the vehicle sunflower oil was performed using a gas chromatography (GC) method with FID detection.
The concentration of C-SAT 080029 in the mixture was quantified using a calibration curve calculated from defined peak areas of the test item. The
analytical method used was developed and validated by LPT for linearity of the calibration curve, accuracy, precision, stability, specificity and
sensitivity.
The measured concentrations ranged from 100.43% to 109.72% of the theoretical value and were well within the admissible limits.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily, 7 days each week for 90 days
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg b.w./day
Basis:
actual ingested
No. of animals per sex per dose:
main study: 10 animals / sex / group in groups 1 to 4; recovery period: 5 animals / sex / group in groups 1 and 4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 7-day dose-range-finding study (LPT Study No. 22830)
- Rationale for animal assignment (if not random): rats were selected because of their proven suitability in toxicology studies and to comply with
regulatory requirements for testing in a rodent animal species
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 6-week recovery period
- Section schedule rationale (if not random):

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: regularly throughout the working day from 7:30 a.m. to 4:30 p.m., on Saturdays and Sundays from 8:00 a.m. to 12:00 p.m. with a
final check at approx. 4:00 p.m.
- Cage side observations checked: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour
patterns


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily


BODY WEIGHT: Yes
- Time schedule for examinations: at the time of allocation of animals to groups, on the day of commencement of treatment and weekly thereafter
always on the same day of the week throughout the experimental period


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:


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 (if drinking water study):
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start of administration and at study termination (main study and recovery period)
- Dose groups that were examined: all dose groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at main study termination (on the first day of dissection); at the end of the recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight)
- How many animals: 10 / sex / group
- Parameters examined: haemoglobin content (HGB), erythrocytes (RBC), leucocytes (WBC), reticulocytes (reti), platelets (PCT), differential blood
count (relative and absolute), haematocrit value (HCT), thromboplastin time (TPT), activated partial thromboplastin time (aPTT), mean corpuscular
volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin (MCHC)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at main study termination (on the first day of dissection); at the end of the recovery period
- Animals fasted: Yes (overnight)
- How many animals: 10 / sex / group
- Parameters examined: albumin, globulin, albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), triglycerides, urea (blood urea nitrogen), 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 main study termination (on the first day of dissection); at the end of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters examined: volume, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin, nitrite
microscopic examination of deposits (epithelial cells, leucocytes, erythrocytes, organisms, further constituents (i.e. sperm, casts), crystalluria)


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in test week 13 approx. 1 - 2 hours after dosing and before any blood sampling
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity


OTHER: Observational screening (righting reflex, body temperature, salivation, startle response, 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, lim rotation and auditory function)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, in all animals of all groups
HISTOPATHOLOGY: Yes, restricted to gropus 1 (vehicle control) and group 4 (high dose)
Statistics:
Students; t-test: all numerical function tests / urinalysis
Multiple t-test based on Dunnett, C.W.: body weight, food consumption, haematology, clinical biochemistry, organ weights (absolute and relative)
Exact test of R.A. Fisher: histopathology

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:
no effects observed
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No test item-related mortality occurred. No test item-related clinical signs of systemic toxicity were noted in the animals treated with
100, 300 or 1000 mg C-SAT 080029/kg b.w./day. The faeces of all the control and test itemtreated animals showed a normal consistency
during the entire treatment period.

BODY WEIGHT AND WEIGHT GAIN
Body weight and body weight gain were not influenced in male and female rats treated with 100, 300 or 1000 mg C-SAT 080029/kg b.w./day.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No feeding study

FOOD EFFICIENCY
No test item-related influence was noted on the food consumption for the male and female rats of all groups treated with the test item.
The visual appraisal of the drinking water consumption revealed no differences between the control and the test item-treated animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No drinking water study.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmological examinations of the ocular structures revealed no lesions of the eyes or the optic region.

HAEMATOLOGY
No test item related influence was noted.

CLINICAL CHEMISTRY
No test item related influence was noted.

URINALYSIS
No test item related influence was noted.

NEUROBEHAVIOUR
The observational and functional screening in test week 13 approximately 1 to 2 hours after application did not reveal any test item-related
changes at any dose level. No influence was noted on fore- and hindlimb grip strength or spontaneous motility.

ORGAN WEIGHTS
The increased liver weights (relative and sbsolute) and kidney weights (absolute) noted in the high dosed male and female rats are considered to be a non-specific change to the high work load of the liver and kidney caused by the high dose level of 1000 mg/kg b.w./day.

GROSS PATHOLOGY
Macroscopic inspection at necropsy did not reveal any changes in the organs and tissues of the rats treated with 100, 300 or 1000 mg CSAT
080029/kg b.w./day.

HISTOPATHOLOGY: NON-NEOPLASTIC
The histopathological examination of the animals treated with 1000 mg C-SAT 080029/kg b.w./day for 90 days did not reveal any morphological
lesions which are considered to be test item-related.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)


HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS

ORGAN WEIGHTS: The increased liver weights (relative and absolute) and kidney weights (absolute) noted in the high dosed male and female rats are considered to be a non-specific adaptive change to the high work load of the liver and kidney caused by the high dose level of 1000 mg/kg b.w./day.


Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption; food efficiency; water consumption and compound intake; ophthalmoscopic examination; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL was > 1000 mg/kg bw.
Executive summary:

The oral toxicity of the test article was tested by daily administration to rats, has been investigated over a period of 13 consecutive weeks.Three groups, each of 10 male and 10 female rats (strain Crl:CD), received the test substance daily by oral gavage (5 ml) at dosages of 100, 300, and 1000 mg/kg/day for a minimum of 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (sun flower oil) and acted as a control.No death occurred during the study. Daily post‑dose observations did not show any significant signs. Detailed clinical signs with neurotoxicity assessment did not show any treatment‑related effects. Neurotoxicity tests and measurements performed at the end of the treatment did not show changes attributable to the test substance. With regard to body weight, no statistically significant differences were observed between control and treated groups. No test item-related influence was observed in food consumption. No findings were seen in the ophthalmic examination performed at the end of the study. No treatment related changes were observed in haematological parameters. No treatment related changes were seen in clinical chemistry parameters. No treatment-related pathological and histopathological changes were seen. No changes were seen in urinalysis. Treatment with 1000 mg/kg bw caused an increase in the absolute and related liver and kidney weights by up to 280 %. 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. Under the present test conditions, the no-observed-adverse-effect-level (NOAEL) for the test article was above 1000 mg/kg bw for systemic changes.