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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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: viscous

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI Wistar rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks at start of treatment
- Weight at study initiation: males 229-261 g, females 177-215 g
- Fasting period before study: no
- Housing: 2 to 3 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 25.6°C
- Humidity (%):28 - 67%
- Air changes (per hr): 15 - 20 per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance was dispersed in propyleneglycol. This dispersion was diluted with tetrahydrofuran (1:2) and directly measured by near infrared (NIR) spectroscopy between 908 and 1676 nm (transmission).
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly

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

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): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment and in week 13
- Dose groups that were examined: all animals before treatment start, animals of control and high dose group in week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Anaesthetic used for blood collection: Yes pentobarbital
- Animals fasted: Yes
- How many animals: all
- Parameters checked in Table 1 below were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked in Table 2 below were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: the night before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table 3 below were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 13
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: meassurement of landing foot splay

IMMUNOLOGY: No

OTHER: oestrous cycles and sperm parameters
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 4 below)

HISTOPATHOLOGY: Yes (see Table 5 below)
Other examinations:
Determination of the oestrous cycles in females of all dose groups during the last two weeks of dosing.

Evaluation of sperm parameters in males of the control and high dose groups at necropsy.
Statistics:
Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System). The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
few incidental findings in animals of all dose groups
Mortality:
mortality observed, treatment-related
Description (incidence):
one animal of the low dose group died after misgavage on Day 90.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the treatment period, there were no adverse changes in the mean body weights or body weight gains between the dose groups either sex. However, slightly reduced body weight gain was noted in males of all dose groups reaching statistical significance compared to the control group. Reduction of body weight gain was not dose dependent and is not considered biologically relevant. see Table 1 Body weight (attached)
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no consistent changes measured in the food consumption during the treatment period between the dose groups. However there was sporadic lower food consumption measurements in Mid dose males and females compared to the Control group with statistical significance, but these were considered unrelated with treatment. see Table 2 Food consumption (attached)
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
see Table 3 Haematology (attached)
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see Table 4 Clinical Chemistry (attached)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed on Week 13.

When compared to the control, lower values were noted in grip strength tests for hind limb in males 1000 mg/kg bw (High dose) however it was not statistically significant.

There was no effect of treatment noted during the assessment of landing foot splay or motor activity.

During evaluation of motor activity, the total travelled distance and the pattern of activity was comparable to the control in both sexes.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see Table 5 Organ weights (attached)
Gross pathological findings:
no effects observed
Description (incidence and severity):
Few findings were observed which were considered incidental or background as these findings were distributed at very low incidence in all dose group animals as well as in control animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Few findings were observed which were considered incidental or background as these findings were distributed at very low incidence in all dose group animals as well as in control animals.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
The oestrous cycles of all females were determined during the last two weeks of dosing. All animals showed the normal distribution of the oestrous phases. There were no differences between dosed and control females.

Sperm parameters were determined after necropsy in control and high dose males. There were no statistically significnat differences in sperm count, sperm motility and not normal sperms.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In this subchronic oral toxicity study the test substance did not exhibit any adverse toxic effects to rats up to and including the highest dose level of 1000 mg/kg/day.