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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:
from 2015-05-19 and 2016-03-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(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:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Age at study initiation: Male animals: 35 – 38 days old Female animals: 35 – 38 days old
- Weight at study initiation: Male animals: 156 – 188 g Female animals: 114 – 130 g
- Housing: 2 or 3 animals of the same sex/ cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 10 – 15 / hour
- Photoperiod: 12 hrs dark / 12 hours light

IN-LIFE DATES: From: 2015-05-20 To: 2015-09-15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in sunflower oil in a frequency based on the stability features of the test item in the vehicle (stable at room temperature at least for one day and at 5 ± 3 °C for 3 days).

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 1.5, 5, 15 mg/mL
- Amount of vehicle: 2 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of formulations (concentration and homogeneity) in the vehicle was performed in the Analytical Laboratory of Test Facility three times during the study. Five samples (5 mL, each) were taken from different places from each concentration (Groups 2, 3 and 4) for analysis of concentration and homogeneity on 3 occasions. Similarly, five samples were taken from different places from the control substance (Group 1) at each occasion and measured. The samples were stored in a refrigerator (at 5 ± 3 °C) until analysis. Formulation samples were diluted with Tetrahydrofuran and with Acetonitrile and analysed by HPLC. Measured concentrations varied between 95 and 106 % of the nominal concentrations and all formulations were considered to be homogeneous.

HPLC conditions:
Detector: 220 nm
Column: Purospher STAR RP-18e 30-2 mm, 3 μm No.: 014664
Mobil Phase: Acetonitrile : water = 60 : 40 (v/v)
Flow Rate: 0.5 mL/min
Injection volume: 5 μL
Temperature: 25 °C
Retention time of CUROX®CC-DC: 4 min
Run time: 6 minutes
Duration of treatment / exposure:
90 or 91 days (depending on the day of necropsy)
Frequency of treatment:
7 days/week basis, every day at a similar time (± 2 hours)
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals/sex in the control and dose groups; 5 animals/sex in the control and high dose groups for recovery observations
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 30, 10 and 3 mg/kg bw/day is based on findings obtained in a previous repeated dose toxicity studies with the test item in the rat (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat OECD 422; GLP, study no. 552.422.2950). Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for selecting satellite groups: five animals per sex in the control and in the high dose group (according to guideline)
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical cage side observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and once weekly thereafter.
- Parameters checked in table [No.1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighing was performed on day 0, then weekly. Fasted body weight was measured on the day of necropsy (Days 90 and 91 for the main groups and Day 118 for the recovery groups).

FOOD CONSUMPTION:
- Food consumption was determined on Day 7, then weekly by reweighing the non-consumed diet in the treatment phase and recovery period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimatization period
- Dose groups that were examined: all control and high dose test animals prior to test termination (day 85)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on main group animals one day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of the recovery period (Day 118)
- Anaesthetic used for blood collection: Yes (Isofluran CP®)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on main group animals one day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of the recovery period (Day 118)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (Day 84)
- Dose groups that were examined: all animals
- Battery of functions tested: sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity

ESTROUS CYCLE: yes
- Time schedule for examinations: during the last two weeks of the treatment period (from Day 77 up to and including Day 90 or 91, depending on the day of the necropsy)
- Dose groups that were examined: all female animals, with exception of the recovery group
- Examinations: A vaginal smear was prepared from each female. The type of cycle (regular or irregular), number of pro-estrous, estrous and diestrous days , number of cycle during the two weeks, number of animals with prolonged diestrous intervals, number of animals with prolonged estrous intervals were evaluated.

SPERM EXAMINATION: yes
- Time schedule for examinations: at necropsy
- Dose groups that were examined: 10 male animals of the control and in 10 male animals of the high dose group
- Quantitative examinations: Testes and epididymides were frozen at necropsy and enumeration was performed on the same animals used later for qualitative examinations. One testis was homogenized for total sperm count determination.
- Qualitative examinations: Sperm motility was determined in a sample of the ductus deferens at necropsy. For the evaluation of the sperm motility, the mean percentage of motile and immotile sperms was determined. The total sperm count and number of immotile sperms were recorded.
A morphological evaluation of ductus deferens sperm sample was performed in the same animals.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, including organ weights (see table No. 4); All animals were necropsied one day after the last treatment (main groups) or after four weeks of recovery (recovery groups).
HISTOPATHOLOGY: Yes (see table No. 5); Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose (30 mg/kg bw/day) groups, including recovery groups. In addition, thymus was also processed histologically in a single animal of the 3 mg/kg bw/day dose group due to macroscopic observations at necropsy.
Statistics:
Statistical analysis was done with SPSS PC+ software package for the following data: body weight, food consumption, estrous cycle, haematology, blood coagulation, clinical chemistry, organ weight data and sperm parameters.
The heterogeneity of variance between groups was checked by Bartlett’s test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of the distribution was not normal, we applied the non-parametric method of Kruskal-Wallis one-way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the two-sample t-test was performed.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings were calculated.
Results were evaluated in comparison to the values of the control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in the section “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p <0.05 and <0.01. Male and female rats were evaluated separately.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
There was no mortality in any group (control, 30, 10 or 3 mg/kg bw/day) during the course of study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test item effect on body weight development was observed in male and female animals at 30 mg/kg bw/day. The body weight changes were only reversible in male animals but not in females. In female animals at 10 mg/kg bw/day, a slight reduction of the mean body weight was also detected. However, the difference was lower than 10 % with respect to the control group. Therefore, it was not considered to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item influence on the mean daily food consumption was detected at 30, 10 or 3 mg/kg bw/day (male or female).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No abnormalities in the eyes of all animals were detected before the treatment and in animals of the high dose group (30 mg/kg bw/day) at termination of the treatment.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no pathological changes in the examined hematological parameters in any of the groups (male and female, 30, 10 or 3 mg/kg bw/day).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No pathologic test item effect was detected at the evaluation of clinical chemistry parameters at 30, 10 or 3 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery did not demonstrate any treatment-related difference in any of the animals in the behaviour or in reactions to different types of stimuli at the end of the treatment period with respect to the controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item related adverse effects on the examined organ weights in male or female animals at 30, 10 or 3 mg/kg bw/day. Statistical differences of some organ weights at 30 mg/kg bw/day in males and females are considered to be partially or fully due to body weight changes in these groups. As these changes are not supported by histopathological alterations, these were considered to be of no toxicological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic findings related to the effect of the test item were not detected in organs or tissues of male or female animals at 30, 10 or 3 mg/kg bw/day at the necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological examination did not reveal alterations related to the test item in the organs or tissues in male or female animals at 30 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
ESTROUS CYCLE
A test item influence on the estrous cycle was not detected at any dose level.

SPERM ANALYSIS
Sperm examinations on sperm cells from the 30 mg/kg bw/day group did not show any test item related effects.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) was determined to be 10 mg/kg bw/d for male and female Wistar rats.
Executive summary:

The objective of this study was to obtain information on the possible health hazards likely to arise from repeated exposure to the test item at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects.

 

The test item was administered orally (by gavage) to Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 30, 10 and 3 mg/kg bw/day corresponding to concentrations of 0, 15, 5 and 1.5 mg/mL, applied in a dose volume of 2 mL/kg bw for 90 or 91 days. 5 animals/ sex in the control and high dose groups assigned to the recovery groups were handled identically up to Day 89 and then observed without administration for another four weeks (recovery observations).

The suitability of the chosen vehicle for the test item and sufficient stability of the test item in the vehicle was analytically verified up front. The test item was stable in the applied concentrations in sunflower oil at room temperature for one day and in a refrigerator (5 ± 3°C) for 3 days.

Concentrations of the test item in the dosing formulations varied from 95 % to 106 % of nominal concentrations at each analytical occasion, thereby confirming proper dosing.

Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at the end of the treatment period. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups, including recovery groups. In addition, the thymus was also processed histologically in single male animals of 3 mg/kg bw/day dose group based on macroscopic observation at necropsy.

The results of study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle only.

No animals died during the course of the study. Toxic signs related to the test item were not detected at any dose level (30, 10 or 3 mg/kg bw/day) at the daily and detailed weekly clinical observations and in the course of the functional observation battery.

The body weight development of male and female animals was reduced in male and female animals at 30 mg/kg bw/day by, which was reversible only in male animals but not in females. Slight reduction of body weight development in female animals at 10 mg/kg bw/day occurred at < 10%. Hence, it was not considered to be toxicologically relevant.

The mean daily food consumption was comparable in animals of the control and test item treated groups (30, 10 and 3 mg/kg bw/day).

There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 30 mg/kg bw/day).

A test item influence on the estrous cycle was not detected (30, 10 and 3 mg/kg bw/day).

Haematology examinations did not reveal test item related toxic changes in the evaluated parameters (30, 10 and 3 mg/kg bw/day). Slight elongation of blood coagulation times (PT, APTT) in male and female animals at 30 mg/kg bw/day were judged to be toxicologically not relevant due to the low degree (mean values remained well within the historical control ranges).

Pathological changes were not detected at the evaluation of clinical chemistry parameters in male or female animals at 30, 10 or 3 mg/kg bw/day. Slightly elevated activity of gamma glutamyl transferase at 30 and 10 mg/kg bw/day (mainly in females) were indicative of a test item influence on the hepatic function as an adaptation response to the altered demand. However, there were no related histopathological changes to substantiate their toxicological relevance.

Specific macroscopic alterations related to treatment with the test item were not observed at the terminal necropsy or at the end of the recovery period.

A test item influence on the examined organ weights was not detected.

Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 30 mg/kg bw/day.

There were no histological lesions related to the test item effect.

 

Under the conditions of the present study, a dose of 30 mg/kg bw/day the test item reduced the body weight development in male and female Hsd.Han:Wistar rats after the consecutive 90-day oral (gavage) administration. Bodyweight changes were reversible in male animals but not in the female animals.

There were no toxicologically relevant changes of the examined parameters in male or female animals at 10 mg/kg bw/day or 3 mg/kg bw/day.

Based on these observations, the No Observed Adverse Effect Level (NOAEL) was determined to be 10 mg/kg bw/day for male and female Hsd.Han:Wistar rats.