Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 04 - June 02 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld ,Germany
- Age at study initiation:Males animals: 57 days
Female animals: 63 days
- Weight at study initiation:Males: 291.1 - 321.6 g
Females: 202.3 - 236.0 g
- Housing: The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm at a room temperature of 22°C ± 3°C (maximum range) and a relative humidity of 55% ± 15% (maximum range).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity: 55% ± 15%
- Air change: 15 to 20 exchanges per hour
- Photoperiod (hrs dark / hrs light): The rooms were lit (about 150 lux at approx. 1.5 metres room height) and darkened for periods of 12 hours each.

IN-LIFE DATES: From: March 04 To: June 02, 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The administration formulations were freshly prepared daily

VEHICLE
- Amount of vehicle (if gavage): 2 mL/kg b.w./day
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Once daily for 90 consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw (total dose)
Dose / conc.:
300 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
10
Control animals:
yes
Positive control:
Not used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at group allocation (test day 9), on test day 1 (before first administration), and once a week thereafter always on the same day of the week throughout the experimental period (test days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90).


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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The drinking water consumption was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed pre-dose (on test day -1) and in test week 13 (test day 90).


HAEMATOLOGY: Yes
- Time schedule for collection of blood:The blood samples were collected at the end of test week 13 (on test day 91, before necropsy)
- Anaesthetic used for blood collection: Yes under isoflrane
- Animals fasted: Yes
- How many animals: All


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:The blood samples were collected at the end of test week 13 (on test day 91, before necropsy)
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:Urine samples were collected from all animals fasted overnight on test day 91 (test week 13).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:At the end of the treatment period (in test week 13), before blood sampling for laboratory examinations), screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli; based on Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment was conducted for all animals. The observations were made 1 to 2 hours after dosing.
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: PATHOLOGY AND HISTOPATHOLOGY
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (see "Any other information on materials and methods")
Statistics:
Toxicology and pathology data were captured, as far as possible, using the departmental computerized systems (Provantis® Integrated preclinical software, version 9.4.0, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups 2 to 4 were compared with the control group 1.
The following statistical methods were used for the data captured with the Provantis system:
Multiple t-test
DUNNETT, C. W.
New tables for multiple Comparisons with a control
Biometrics, 482-491 (Sept 1964)
Exact test of R. A. FISHER Histology

The following statistical methods were used for the data not captured with the Provantis system:
STUDENT's t-test



Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was observed on the body weight and the body weight gain of the male and female animals repeatedly treated orally with 100, 300 or 1000 mg Oxooil LS13/kg b.w./day compared to the control animals throughout the course of the study.
No test item-related differences were noted for the body weight at autopsy between the test item-treated animals and the control animals
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
A slightly, but statistically significantly (at p ≤ 0.05 or p ≤ 0.01) increased food consumption was noted for group 3 (intermediate dose) and group 4 (high dose) in a few test weeks. However, the marginal changes are considered as coincidental and not test item-related.
The statistically significant differences to the control group's food consumption considered not to be test item-related .
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was observed on the haematological and coagulation parameters for the male and female animals treated orally with 100, 300 or 1000 mg Oxooil LS13/kg b.w./day compared to the control animals on test day 91.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the biochemical parameters of the male and female animals treated with 100, 300, or 1000 mg Oxooil LS13/kg b.w./day on test day 91.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes were noted for the specific gravity and the pH value of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.
A few slight but statistically significant (at p ≤ 0.01 or p ≤ 0.05) changes were noted for the pH value of the urine of the male and/or female animals at various dose levels in comparison to the control. However, the slight decrease of up to 9% is considered as coincidental and not test item-related.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 100, 300 or 1000 mg Oxooil LS13/kg b.w./day in test week 13.
A statistically significant (at p ≤ 0.05) increase by 0.3°C was noted for the body temperature of the female animals treated with 300 mg Oxooil LS13/kg b.w./day compared to the control group in test week 13. However, the marginal change is considered as coincidental and not test item-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related changes were noted for the relative and absolute organ weights of the animals treated orally with 100 mg Oxooil LS13/kg b.w./day at terminal sacrifice on test day 91.
Starting at the intermediate dose of 300 mg Oxooil LS13/kg b.w./day, statistically significantly (at p ≤ 0.05 or p ≤ 0.01) increased kidney weights were noted for the male animals, and increased liver weights were noted for the male and female animals. The changes in organ weights are considered to be test item-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic inspection at necropsy did not reveal any test item-related changes in the organs and tissues of the animals treated orally with 100, 300 or 1000 mg Oxooil LS13/kg b.w./day at terminal sacrifice on test day 91 or 92.
A few minor macroscopic findings were noted in form of an enlarged pituitary gland for two female low dose animals and one female high dose animal, or a haemorrhagic focus with a diameter of approx. 2 mm in the mucosa of the stomach for one female intermediate dose animal. However, these changes are not considered to be test item-related but to be normal spontaneous background changes due to their isolated occurrence and the lack of dose-relationship.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Morphological lesions considered to be related to the test item Oxooil LS13 were noted in the kidneys of the male animals, and the stomach of the male and female animals at the high dose of 1000 mg Oxooil LS13/kg b.w./day. Individual male and/or female animals of the control group 1 and the low and intermediate dose groups 2 and 3, revealed a few findings in the kidneys and/or the stomach that were similar to those noted for the high dose animals, but with clearly lower incidences and severities, and never being statistically significant compared to the control.
The test item related changes in the kidneys of the male rats are classified as Alpha 2u-globulin nephropathy.
Further, 4 of 10 male high dose animals revealed a hepatocyte hypertrophy that did not achieve statistical significance but corresponds to the slight increase noted for the relative and absolute liver weights , and therefore is considered to be test item-related, too.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw (total dose)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw (total dose)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, the experimental no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS13/kg b.w. by daily oral administration, in particular, as no test item-related changes were noted at histopathological examination.