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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:
2013/2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline tudy + GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Oxooel 9N
- Physical state: liquid / colorless, clear
- Analytical purity: The IR spectrum shows the expected bands. The 1H-NMR spectrum of the test item shows the signals expected for the given chemical compound
- Lot/batch No.: 93932509T0
- Expiration date of the lot/batch: 18.09.2015
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor and the sponsor holds this responsibility.
- Storage condition of test material: room temperature, under N2

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42+/- 1 days
- Housing: animals were housed together (5 animals per cage) in H-Temp polysulfonate cages type 2000P
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

The stability of Oxooel 9 N in corn oil at room temperature for a period of 7 days was proven before the start of the administration period.
The test-substance preparations were produced at least weekly and stored at room temperature. The administration volume was 4 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: limit test



Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations: yes, daily

DETAILED CLINICAL OBSERVATIONS: Yes
prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: weekly intervals

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily visual inspection of the water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to administration and at day 91
- Dose groups that were examined: 0 and 1000 mg/kg/d

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals:all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the administration period
- Metabolism cages used for collection of urine: Yes


FUNCTIONAL OBSERVATION BATTERY Yea
- Time schedule for examinations: at the end of administration period
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity /home cage observation / open field observation

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Sperm parameters:
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals. Sperm motility examinations were carried out in a randomized sequence. Sperm head count (testis and cauda epididymis) and morphology were evaluated in all groups.
Estrous cycle:
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.
Statistics:
DUNNETT's test (two-sided), one-way analysis using KRUSKALWALLIS test (two-sided)

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
One female animal of test group 1 (100 mg/kg bw/d) was sacrificed in a moribund condition on study day 77. Upon histological analysis, no findings were noted that could explain the cause of this moribund state.

BODY WEIGHT AND WEIGHT GAIN
No significant and test substance-related changes of mean body weights in both sexes were observed. The same was true for mean body weight change values. For male animals of test group 2 (300 mg/kg bw/d) mean body weight change value was significantly higher on study day 14 (+9.4%). This single event was assessed as spontaneous in nature and not to be of toxicological relevance.

CLINICAL EXAMINATION
Salivation after treatment from slight to moderate was observed in all male and female animals of test groups 2 and 3 (300 and 1000 mg/kg bw/d) as well as in 6 of 10 male and 1 of 10 female animals of test group 1 (100 mg/kg bw/d). From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract:
On study days 76 and 77, one female animal of test group 1 (100 mg/kg bw/d) showed poor general condition, paresis of both hindlimbs and apathetic behavior. These findings were not related to the test substance. One female animal of test group 3 (1000 mg/kg bw/d) had a swollen eyelid between study days 87 to 93. The finding was assessed as being incidental and not related to treatment.

WATER AND FOOD CONSUMPTION
No test substance-related findings were observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings were observed.
One female animal of test group 3 (1000 mg/kg bw/d) had a swollen eyelid between study days 87 to 93. Therefore, on the day of examination the animal was not able to open the eyelid completely. The finding was assessed as being incidental and not related to treatment. In addition, all other apparent findings were assessed as being incidental in nature since they occurred in the control group and did not show a dose-response relationship.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related adverse changes among clinical chemistry parameters were observed.
At the end of the administration period in males of test groups 1 and 3 (100 and 1000 mg/kg bw/d) alanine aminotransferase (ALT) activities were decreased. Creatinine levels were increased in males of test group 2 (300 mg/kg bw/d), but decreased in females of the same test group. Urea values were decreased in females of test groups 1 and 2 (100 and 300 mg/kg bw/d). However, all mentioned parameters were not dose-dependently altered and, therefore, these changes were regarded to be incidental and not treatment-related.
In males of test groups 2 and 3 (300 and 1000 mg/kg bw/d) total bilirubin levels were decreased and potassium levels were increased. The decrease of bilirubin values without any signs of anemia was most probably due to an increased conjugation rate in the liver and an accelerated excretion via the bile. This effect was regarded as adaptive and not adverse. Potassium was the only changed electrolyte in these individuals. Therefore, this alteration was regarded as treatment-related, but not adverse.

URINALYSIS
No treatment-related adverse changes among urinalysis parameters were observed.
In male animals of test groups 2 and 3 (300 and 1000 mg/kg bw/d) the incidences of granulated and epithelial cell casts and transitional epithelial cells and, additionally, in males of test group 1 (100 mg/kg bw/d) the incidence of casts were higher compared to controls. As shown in the histopathological investigation these findings were due to the “inducible α2u globulin nephropathy syndrome” which is described as species- and gender-specific effect in male rats (G. C. Hard et al., 1993). Therefore, the finding is not relevant as adverse effect for humans.
In males of test group 2 (300 mg/kg bw/d) higher incidences of blood were found in the urine. This was due to four individuals with blood values grade 2 and 3. Blood grade 2 was only found in one male of test group 3 (1000 mg/kg bw/d). This effect was regarded as not dose-dependent and, therefore, as being incidental and not treatment-related.


FOB
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Regarding the single and overall motor activity, no test substance-related deviations to the control were noted for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).

ORGAN WEIGHTS
Statistically significant deviations of the absolute weight in adrenal glands and brain as well as the relative weight decrease of the cuada epipidymis in males occurred without dose dependency. Thus, they were not considered to be treatment-related. The absolute weight increases of the thyroid gland were all below the maximal historical control value (28.8 g) and occurred without concurrent relative weight increased. Therefore, they were regarded as incidental. The statistically significant absolute and relative weight increase in the kidneys of males had a histopathological correlate and was considered to be treatment-related. The statistically significant absolute and relative liver weight increase in males and females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) were both above the maximal historical control range values (absolute/relative, male: 9.261 g / 2.39%, female: 5.771 g / 2.723%) and were regarded as treatment-related.


GROSS PATHOLOGY
All gross findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were observed in the kidneys of males and in the liver and thyroid glands of male and females.
When compared with the control group 0, a dose-dependent increased accumulation and dissemination of eosinophilic droplets was observed in the proximal convoluted tubules of male test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d). Eosinophilic droplets were positive in the Mallory-Heidenhain stain and in the immunhistochemical stain with an antibody to α2uglobulin. In test group 3, only one male showed minimal granular casts. Multifocal basophilic tubules increased rather in incidence than grading when compared with the control group, but without a clear dose-dependent relationship.
A dose dependent diffuse hepatocytic hypertrophy was seen in males and females starting from test group 2 (300 mg/kg bw/d).
Hypertrophy/hyperplasia in males and females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) was regarded as treatment-related. Altered colloid was considered to be treatmentrelated in males of test group 2 and 3 (300 and 1000 mg/kg bw/d) and in females of the high dose (1000 mg/kg bw/d).
All other findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.


ESTROUS CYCLE
No test substance-related effects on estrous cycle length and the number of cycles were obtained.

SPERM PARAMETERS
Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

Effect levels

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: No treatment-related, adverse effects were observed up to limit dose

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion