Registration Dossier

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:
2015-12-22 and 2016-08-19
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
Details on species / strain selection:
The rat is regarded as a suitable species for toxicity studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in toxicity studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 43 – 48 days old (males/females)
- Weight at study initiation: 162 – 204 g (males), 115 – 144 g (females)
- Housing: 5 animals of the same sex/ cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: The food and drinking water is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The analytical certificate for the batch used in the study is presented in the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 12
- Humidity (%): 30 - 70
- Air changes (per hr): 10

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 the vehicle (sunflower oil). Formulations were prepared in the formulation laboratory of Test Facility beforehand not longer than for three days and stored at 5 ± 3°C until use.

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. The suitability of the chosen vehicle for the test item was analytically proven.
- Concentration in vehicle: 500 mg/mL, 150 mg/mL and 50 mg/mL
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: 1506-4604
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.
Measured concentrations varied between 95 and 107 % of the nominal concentrations and all formulations were considered to be homogeneous. The suitability of the chosen vehicle for the test item was analytically proven. Recovery was between 97 and 98 % of nominal concentrations at 25 and 500 mg/mL in sunflower oil, respectively. The test item proved to be stable at room temperature at least for 4 hours and at 5 ± 3°C for 3 days. Samples were analyzed via HPLC method.
Duration of treatment / exposure:
90 or 91 days (depending on the day of necropsy)
Frequency of treatment:
7 days/week at a similar time
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 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 1000, 300 and 100 mg/kg bw/day is based on findings obtained in a previous repeated dose toxicity studies with the test item in the Rat (28-day Oral Toxicity Study in the Rat, Report no. 04/915-100P; OECD 407; GLP). 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: According to the requirements of OECD Guideline 408.
- Post-exposure recovery period in satellite groups: 4 weeks (28 days)
Positive control:
not required

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: on Day 0 and then weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- The 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 acclimation period
- Dose groups that were examined: on all control and high dose test animals prior to test termination (Day 88)

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
- 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: in all animals except one female at 300 mg/kg bw/day due to sample destruction
- Parameters checked in table No. 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (Day 90)
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength and motor activity

IMMUNOLOGY: No

OTHER:

ESTROUS CYCLE
- 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
- Examinations: A vaginal smear was prepared from each female. The type of cycle (regular or irregular), number of days in pro-estrous, estrous and diestrous, number of cycle during the two weeks, number of animals with prolonged diestrous, number of animals with prolonged estrous were evaluated.

SPERM EXAMINATION
- Time for examinations: at necropsy
- Dose groups that were examined: 10 animals of the control and 10 animals at high dose group
- Quantitative examinations: Testes and epididymides were frozen at the necropsy and enumeration was performed later. The total number of homogenization of one side testis was enumerated.
- Qualitative examinations: Sperm motility was determined from sample of ductus deferens at the 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 sperms 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 period (recovery groups).

HISTOPATHOLOGY: Yes (see table No. 5); Full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups, including recovery groups. In addition, spleen was subjected to histopathological processing in female animals at 300 mg/kg bw/day group due to macroscopic and histological observations at 1000 mg/kg bw/day.
Statistics:
Statistical analysis was done for the following data: body weight, food consumption, estrous cycle, hematology, blood coagulation, clinical chemistry, organ weight data and sperm parameters.
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance 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, and pathology and histopathology findings was 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:
effects observed, treatment-related
Description (incidence and severity):
Test item related salivation was observed in male and female animals of 1000 mg/kg bw/day and in male animals of 300 mg/kg bw/day, which however was not considered to be adverse because salivation appeared shortly after the administration of the test item and ceased within one hour thereafter.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences with respect to the control were detected at the slightly lower mean body weight of male animals at 100 mg/kg bw/day from Day 63 up to and including Day 89. The differences were with low degree – ≥-7 % relative to the control group – and similar findings were not detected at the higher doses therefore these were not considered to be toxicologically relevant.
Sporadic statistical significances were noted for the slightly lower mean body weight gain of male animals at 1000 mg/kg bw/day (between Days 77 and 84), at 300 mg/kg bw/day (between Days 0 - 7 and 77 - 84) and at 100 mg/kg bw/day (between Days 14-21, 28-35 and 77-84). These slight changes had no influence on the mean summarized body weight gain (from Day 0 to Day 89) at 1000 or 300 mg/kg bw/day. The mean summarized body weight gain was slightly lower than in the control group only in male animals at 100 mg/kg bw/day therefore it was judged to be indicative of biological variation and not related to the test item as similar findings were not detected at the higher doses.
Similarly, the mean body weight gain of female animals at 1000 mg/kg bw/day remained below the relevant control with statistical significance between Days 84 and 89. This slight change had no significant influence on the mean body weight or on summarized body weight gain of this group. Therefore, this slight change in mean body weight gain was judged to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The daily mean food consumption was reduced with respect to the controls in male animals at 100 mg/kg bw/day from week 2 up to and including week 13 in compliance with the body weight gain and body weight values of this group. The differences were with low degree – ≥ -16 % relative to the control group – and similar findings were not detected at the higher doses therefore these were not considered to be toxicologically relevant.
Some statistically significant differences were detected at the lower mean daily food consumption in male animals at 1000 mg/kg bw/day on week 1 and at 300 mg/kg bw/day on weeks 8 and 9. Similarly, statistical significances were noted for the slightly lower mean daily food consumption of female animals at 1000, 300 and 100 mg/kg bw/day on weeks 8 and 9. This slight reduction in the food consumption was not considered to be biologically or toxicologically meaningful because of the sporadic occurrence and of small difference to the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological investigations revealed test item related slight elevation in the white blood cell count and in the percentage of reticulocytes in female animals at 1000 mg/kg bw/day at termination of the treatment. Slight reduction was also observed in the percentage of eosinophilic granulocytes in female animals at 1000 mg/kg bw/day. The differences with respect to the control partially of fully recovered after the four weeks post-treatment period. The changed parameters were in correlation with the splenic changes of high dose treated female animals (macroscopic, organ weight and histopathology).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Elevated activity of aspartate aminotransferase and elevated concentration of total bilirubin were observed in female animals at 1000 mg/kg bw/day as a test item related adaptive changes. These changes were in correlation with splenic changes (macroscopic, organ weight and histopathology) in female animals administered with high dose.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The spleen weights were significantly elevated in female animals at 1000 mg/kg bw/day at the termination of the treatment period. Minor differences with respect to the control were detected in female animals at 1000 mg/kg bw/day at the end of the recovery period. However there were no related histopathological alterations in the spleen tissue therefore spleen weight changes was considered to be reversible.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic finding – enlarged spleen – related to the effect of the test item was detected in female animals at 1000 mg/kg bw/day at the necropsy.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyperplasia of spleen – in the red pulp (the erythroid, myeloid cells and the megakaryocytes) and in the white pulp (the peri-arteriolar lymphoid sheath, the number and size of lymphoid follicles and the size of marginal zones) – was detected in female animals at 1000 mg/kg bw/day at the termination of the treatment. Splenic hyperplasia was reversible as it was not present in high dose treated female animals at the end of the treatment period. Lymphocytic and histiocytic infiltration was observed in the liver, in some portal triads in one male animal in some female. This phenomenon could be in connection with the splenic hyperplasia and could be considered as the consequence of the possible stimulative impact of the 1000 mg/kg bw/day dose of test item as well.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
MORTALITY
There was no mortality in any group (control, 1000, 300 or 100 mg/kg bw/day) during the course of study.

CLINICAL OBSERVATIONS
- Daily clinical observations: Adverse signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations.
Test item related salivation was observed in male and female animals at 1000 mg/kg bw/day and only in male animals at 300 mg/kg bw/day. The behavior and physical condition of animals in 1000, 300, and 100 mg/kg bw/day and in the control group were considered to be normal during the entire observation period.
Salivation appeared in male animals in a dose related manner (13/15 at 1000 mg/kg bw/day and 3/10 at 300 mg/kg bw/day) regarding the onset, and duration. In female animals, salivation was detected in a lower incidence (7/15) at 1000 mg/kg bw/day only. Control male and female animals did not salivate therefore salivation was considered to be related to the treatment with the test item. Salivation was seen shortly after the daily administration of the test item and it ceased shortly thereafter therefore was judged to be not adverse. There were no clinical signs in male or female animals of control and high dose group during the four week post-treatment period.

- Weekly detailed clinical observations: The general physical condition and behavior of animals were considered to be normal at the detailed weekly observations throughout the entire observation period (treatment and recovery periods).
There were no clinical signs, changes in the behavior or autonomic activity in any of the animals at 1000, 300 or 100 mg/kg bw/day or in the control group at the detailed weekly clinical observations.

BEHAVIOUR (FUNCTIONAL FINDINGS)
Functional observation battery did not demonstrate any treatment-related difference in any of the animals in the behavior or in reactions to different types of stimuli at the end of the treatment period with respect to the controls (1000, 300 or 100 mg/kg bw/day).
The behavior and reactions to different types of stimuli or manipulations of the animals were considered to be normal in the control and all test item treated groups at the functional observations.

WEIGHT ASSESSMENT
The body weight and body weight gain of the male and female animals were unaffected in all test item treated groups (1000, 300 or 100 mg/kg bw/day) throughout the three month treatment period.
The mean body weight was similar in male and female animals in the control and 1000 or 300 mg/kg bw/day treated groups and in female animals at 100 mg/kg bw/day during the entire treatment period.
Statistically significant differences with respect to the control were detected at the slightly lower mean body weight of male animals at 100 mg/kg bw/day from Day 63 up to and including Day 89. The differences were with low degree – ≥-7 % relative to the control group – and similar findings were not detected at the higher doses therefore these were not considered to be toxicologically relevant.
Sporadic statistical significances were noted for the slightly lower mean body weight gain of male animals at 1000 mg/kg bw/day (between Days 77 and 84), at 300 mg/kg bw/day (between Days 0 - 7 and 77 - 84) and at 100 mg/kg bw/day (between Days 14-21, 28-35 and 77-84). These slight changes had no influence on the mean summarized body weight gain (from Day 0 to Day 89) at 1000 or 300 mg/kg bw/day. The mean summarized body weight gain was slightly lower than in the control group only in male animals at 100 mg/kg bw/day therefore it was judged to be indicative of biological variation and not related to the test item as similar findings were not detected at the higher doses.
Similarly, the mean body weight gain of female animals at 1000 mg/kg bw/day remained below the relevant control with statistical significance between Days 84 and 89. This slight change had no significant influence on the mean body weight or on summarized body weight gain of this group. Therefore, this slight change in mean body weight gain was judged to be toxicologically not relevant.
The mean body weight and body weight gain were similar in male and female animals in the control and 1000 mg/kg bw/day groups throughout the recovery period.

FOOD CONSUMPTION
There were no test item or treatment related changes in the food consumption of male and female animals at any dose level (1000, 300 and 100 mg/kg bw/day). The daily mean food consumption was reduced with respect to the controls in male animals at 100 mg/kg bw/day from week 2 up to and including week 13 in compliance with the body weight gain and body weight values of this group. The differences were with low degree – ≥ -16 % relative to the control group – and similar findings were not detected at the higher doses therefore these were not considered to be toxicologically relevant.
The daily mean food consumption of test item treated male animals at 1000 or 300 mg/kg bw/day and of female animals at 1000 or 300 or 100 mg/kg bw/day was comparable to the controls during the three month treatment period. However, some statistically significant differences were detected at the lower mean daily food consumption in male animals at 1000 mg/kg bw/day on week 1 and at 300 mg/kg bw/day on weeks 8 and 9. Similarly, statistical significances were noted for the slightly lower mean daily food consumption of female animals at 1000, 300 and 100 mg/kg bw/day on weeks 8 and 9. This slight reduction in the food consumption was not considered to be biologically or toxicologically meaningful because of the sporadic occurrence and of small difference to the control group. The mean daily food consumption of male and female animals was similar in the control and 1000 mg/kg bw/day during the recovery period.

OPHTHALMOLOGICAL EXAMINATION
The eyes were without any abnormalities in all animals before the treatment and in animals in the control and high dose groups at termination of the treatment.

HEMATOLOGY
Hematological investigations revealed test item related slight elevation in the white blood cell count and in the percentage of reticulocytes in female animals at 1000 mg/kg bw/day at termination of the treatment. These changes were in correlation with splenic changes (macroscopic, organ weight and histopathology) in female animals administered with high dose. Slight reduction was also observed in the percentage of eosinophilic granulocytes in female animals at 1000 mg/kg bw/day. The differences with respect to the control partially or fully recovered after the four weeks post-treatment period.
Slight but statistically significant differences with respect to the control were detected in male animals at the lower mean percentages of the monocytes (MONO) and reticulocytes (RET) at 100 mg/kg bw/day. The prothrombin time (PT) was slightly longer than in the control group in male animals at 100 and 1000 mg/kg bw/day
In the female animals at 1000 mg/kg bw/day, the mean white blood cell count (WBC) and mean percentages of basophilic granulocytes (BASO) and reticulocytes were statistically significantly elevated while the mean percentage of eosinophilic granulocytes (EOS) and mean platelet count (PLT) were reduced compared to the control animals. On the contrary, the mean platelet count was higher than in the control group in female animals at 100 mg/kg bw/day.
Elevation of the white blood cell count and percentage of reticulocytes was significant in those female animals at 1000 mg/kg bw/day, in which splenic changes were detected at necropsy (enlargement, increased weight) or at histopathological examination (splenic hyperplasia).
Recovery groups
In the female animals of recovery group at 1000 mg/kg bw/day, the mean percentage of eosinophilic granulocytes was lower while the mean percentage of reticulocytes was higher than those in the control group. These differences with respect to the control group were less than at the end of the treatment period. Moreover values remained well within the historical control range, therefore were considered to be reversible.

CLINICAL CHEMISTRY
Elevated activity of aspartate aminotransferase and elevated concentration of total bilirubin were observed in female animals at 1000 mg/kg bw/day as a test item related adaptive changes. These changes were in correlation with splenic changes (macroscopic, organ weight and histopathology) in female animals administered with high dose.
In male animals, statistically significant differences with respect to the control were detected at the lower mean concentrations of total bilirubin (TBIL) at 1000 mg/kg bw/day and sodium (at 1000 and 300 mg/kg bw/as well as at the higher mean concentration of glucose (GLUC) at 300 mg/kg bw/day at the end of the treatment period.
In the female animals, statistically significant differences with respect to the control were detected at the higher mean activity of aspartate aminotransferase (AST) and higher mean concentration of total bilirubin at 1000 mg/kg bw/day and at the higher mean concentration of chloride (Cl-) at 300 mg/kg bw/day.
In male animals of the 1000 mg/kg bw/day recovery group, the mean glucose concentration was slightly lower than in the control group.
In female animals at 1000 mg/kg bw/day recovery group, the mean concentrations of urea and inorganic phosphorous (Pi) remained below the control group.
These sporadic statistical differences (GLUC, Pi, Na+ and Cl-) were considered to be of little or no biological relevance. Although the mentioned differences between the control and test item treated groups were statistically significant, these were small and all values were within of the historical control ranges for these parameters. Moreover, some of these differences were observed in the lower dose groups but not in the high dose group (GLUC in male animals and Cl- in female animals, each at 300 mg/kg bw/day). Therefore, these findings were not considered to be of any toxicological relevance.

NECROPSY
Macroscopic finding – enlarged spleen – related to the effect of the test item was detected female animals at 1000 mg/kg bw/day at the necropsy.
There were no macroscopic changes in the organs and tissues of male animals at 1000, 300 or 100 mg/kg bw/day or in animals in the control group at the termination of the treatment period.
Significantly enlarged spleen (6-7 cm long) was observed in several female animals at 1000 mg/kg bw/day (6/10). Slight or moderate hydrometra occurred in several female animals in the control (4/10), 100 mg/kg bw/day (2/10) and 300 mg/kg bw/day (2/10) groups.
In the recovery group of 1000 mg/kg bw/day, smaller than normal testes and epididymides were observed in one male animal (1/5) at the terminal necropsy. There were no macroscopic changes in the control male animals observed for four weeks after the termination of the treatment.
Dilated uterus (2/5) and uterine horns (1/5) with thickened wall were observed in high dose recovery female animals at the end of the four weeks observation period. In the female control recovery group, unopened vagina and dilated uterus with purulent content were noted for one female control animal (1/5) and slight hydrometra for another one (1/5).

ORGAN WEIGHT
Spleen weight changes were noted for female animals at 1000 mg/kg bw/day at the termination of the treatment period. The changes in the spleen weights were recovered by the end of post-treatment period.
The mean fasted body weight and mean kidney weights were slightly lower compared to the control group in male animals at 100 mg/kg bw/day. Statistically significant differences with respect to the control were noted for the higher mean organs weights relative to body weight in male animals as follows: adrenal gland at 1000 mg/kg bw/day; brain, spleen, testes, epididymides and adrenal glands at 300 mg/kg bw/day; brain, heart, testes and epididymides at 100 mg/kg bw/day. These minor changes in organ weights relative to body weight were mainly attributed to slightly reduced fasted mean body weight of test item treated animals. There were no dose relevancy and related histological alterations therefore these were judged to be toxicologically not relevant.
As a consequence of the slight changes in fasted body weight, statistical significance was detected at the lower mean fasted body weight referred to the brain weight in male animals at 300 and 100 mg/kg bw/day.
In the female animals, the mean weights of spleen (absolute and relative to body and brain weights) and of adrenal glands (relative to the body and brain weights) were significantly elevated with respect to the control at 1000 mg/kg bw/day. The mean weight of thymus and uterus (absolute and relative to the brain weight, both) were slightly but statistically lower compared to the control group at 300 mg/kg bw/day.
At the end of the recovery period, slightly higher and statistically significant spleen weights (absolute and relative to body and brain weights) compared to the control was noted in male animals at 1000 mg/kg bw/day. Similar findings were not detected in male animals at the termination of the treatment period and related histopathological changes were not seen in the spleen of animals of main group or recovery groups. The changes in spleen weights of male recovery animals were with small degree and were not considered to be related to the test item effect.
In the female recovery group treated with the test item at a dose of 1000 mg/kg bw/day, the mean spleen weight referred to the body weight slightly exceeded that in the control group. However the differences with respect to the control were minor, there were no related histopathological alterations in the spleen tissue therefore spleen weight changes was considered to be reversible.

HISTOPATHOLOGY
Histological examination revealed hyperplasia of spleen in female animals at 1000 mg/kg bw/day. This splenic hyperplasia was detected in the red pulp (the erythroid, myeloid cells and the megakaryocytes) and in the white pulp (the peri-arteriolar lymphoid sheath, the number and size of lymphoid follicles and the size of marginal zones). Based on the cytomorphology and the distribution of these cells and comparing to the intact normal splenic structure, the neoplastic diseases were excluded.
Hematopoiesis (extramedullary erythroid and myeloid hematopoiesis) in the red pulp is normally present in rats and other rodents and is characterized by scattered, poorly demarcated foci of cells with intensively basophilic nuclei. Megakaryocytes (multinucleated giant cells) are also normally present in the red pulp in rats. Hyperplasia of erythroid and myeloid hematopoietic tissues in the splenic red pulp is indicated by the enlargement of number and size of these cell groups and the number of megakaryocytes. Lymphoid hyperplasia (hyperplasia of the white pulp) may be focal or diffuse. The white pulp includes peri-arteriolar lymphoid sheets (PALSs), primary and secondary lymphoid follicles and a marginal zone.
A careful comparison of the spleen of the treated and control animals (the PALSs, the size and maturation of lymphoid follicles, the presence or absence of the marginal zone cells and the relative number of smaller lymphoid aggregates scattered thought the spleen) was performed in understanding the specific stimulative impact of the test article (Haley, 2013). The splenic hyperplasia was observed in a proportion of female animals (6/10) belonging to the 1000 mg/kg bw/day group and was not detected in the recovery group, therefore it was considered to be reversible changes. This phenomenon could be in connection with a possible stimulative impact of the 1000 mg/kg bw/day dose of the test item in some more susceptible female animals.
No lesions were detected in the other investigated hematopoietic organs and tissues (including the bone marrow, the thymus, the different lymph nodes and the different lymphoid tissues, for example the bronchus associated and gut associated lymphoid tissues) of the treated animals..
In addition, lymphocytic and histiocytic infiltration was observed in some portal triads in the liver in some female (6/10) and in one high dose treated male animal (1/10) subjected to necropsy at the end of the treatment period. This phenomenon probably was in connection with the splenic hyperplasia in female animals and could be considered as the consequence of the possible stimulative impact of the 1000 mg/kg bw/day dose of test item. Based on the cytomorphology and the localization of these cells the neoplastic origin was excluded.
Pulmonary alveolar emphysema was observed in some animals in the control (1/10 male and 1/10 female) and in 1000 mg/kg bw/day (1/10 male) groups. Pulmonary emphysema was in connection with the hypoxia, dyspnea and circulatory disturbance, developed during the exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs occurred sporadically (3/10 male and 2/10 female in control group; 2/10 male and 1/10 female at 1000 mg/kg bw/day) and was considered as a physiological, immuno-morphological phenomenon.
In one female control animal (1/10) focal alveolar histiocytosis was observed in the lungs. Alveolar histiocytosis is a common incidental finding in older rats and consists of small collections of alveolar macrophages with abundant foamy (lipid-containing) cytoplasm.
Dilatation of uterine horns was observed in some female control (4/10) animal. Dilatation of uterine horns without inflammation or other pathological lesions is a slight neuro-hormonal phenomenon in connection with the sexual function –pro-estrus phase- of the inner genital organs.
In one control female animal pyelitis (1/10) was detected in the kidney as an individual disease.
In the recovery animals, alveolar emphysema (2/5 male control; 1/5 female at 1000 mg/kg bw/day) and hyperplasia of bronchus associated lymphoid tissue (1/5 female at 1000 mg/kg bw/day) were detected in the lungs.
Dilatation of uterine horns was detected in some female animals in control (1/5) and 1000 mg/kg bw/day (3/5) groups at the end of the recovery period.
Individual disorders were observed in the genital organs of one male animal at 1000 mg/kg bw/day – (1/5) decreased intensity of spermatogenesis in the testicles and lack of mature spermatozoa in the epididymis– and in one control female animal – (1/5) purulent endometritis – at the end of the recovery period.
These findings are common also in untreated experimental rats without toxicological significance. No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the alimentary system, the pancreas, the cardiovascular system, the skeleton, the muscular system, or the central and peripheral nervous system was observed. The histological picture and cyto-morphology of endocrine glands and tissues were the same in the control and treated animals.

SPERM EXAMINATIONS
Sperm examinations (quantitative or qualitative) on sperm cells from the 1000 mg/kg bw/day group did not show any test item related changes.
Slightly higher mean percentage of motile sperm cells and slightly lower mean percentage of immotile sperms at 1000 mg/kg bw/day respective to control were statistically significant.
The mean total sperm count, the mean percentage of sperms with normal or abnormal (separated head and tail) morphology were similar in animals at 1000 mg/kg bw/day and in the control group.

EXAMINATION OF ESTROUS CYCLE
A test item influence on the estrous cycle was not detected at any dose level.
Statistical or biological significances with respect to the control were not observed in the examined parameters of estrous cycle (mean number of cycle, mean length of cycles, or percentage of animals with regular cycle or irregular estrous cycle, number of days in pro-estrus, estrus or diestrus, percentage of animals with prolonged estrous or diestrus) at 1000, 300 or 100 mg/kg bw/day. The number and percentage of animals with regular cycles were the highest at 1000 mg/kg bw/day while animals with irregular estrous cycles were reduced at 1000 mg/kg bw/day in comparison to the control group.

Effect levels

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Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
spleen
Treatment related:
yes
Dose response relationship:
no

Applicant's summary and conclusion

Conclusions:
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL: 1000 mg/kg bw/day for male Hsd.Han:Wistar rats
NOAEL: 300 mg/kg bw/day for female Hsd.Han: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 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), 1000, 300 and 100 mg/kg bw/day corresponding to concentrations of 0, 500, 150 and 50 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 test item in the vehicle was analytically verified up front. Test item was stable in the applied concentrations in sunflower oil at room temperature for four hours and in a refrigerator (5 ± 3°C) for 3 days. Concentrations of the test item in the dosing formulations varied from 95 % to 107 % 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 spleen was also processed histologically in all female animals of 300 mg/kg bw/day dose group based on macroscopic and histopathology findings in spleen at 1000 mg/kg bw/day to facilitate a better estimating of NOAEL. 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 (1000, 300 or 100 mg/kg bw/day) at the daily and detailed weekly clinical observations and in the course of the functional observation battery. Test item related salivation was observed in male and female animals of 1000 mg/kg bw/day and in male animals of 300 mg/kg bw/day, which however was not considered to be adverse because salivation appeared shortly after the administration of the test item and ceased within one hour thereafter. No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level during the treatment or recovery periods. The body weight development of male and female animals was undisturbed in the course of the entire study. The mean daily food consumption was comparable in animals of the control and test item treated groups (1000, 300 and 100 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 1000 mg/kg bw/day). A test item related influence on the estrous cycle was not detected (1000, 300 and 100 mg/kg bw/day). Hematological investigations revealed test item related slight elevation in the white blood cell count and in the percentage of reticulocytes in female animals at 1000 mg/kg bw/day at termination of the treatment. Slight reduction was also observed in the percentage of eosinophilic granulocytes in female animals at 1000 mg/kg bw/day. The differences with respect to the control partially of fully recovered after the four weeks post-treatment period. The changed parameters were in correlation with the splenic changes of high dose treated female animals (macroscopic, organ weight and histopathology). Elevated activity of aspartate aminotransferase and elevated concentration of total bilirubin were observed in female animals at 1000 mg/kg bw/day as a test item related adaptive changes. These changes were in correlation with splenic changes (macroscopic, organ weight and histopathology) in female animals administered with high dose. Macroscopic finding – enlarged spleen – related to the effect of the test item was detected in female animals at 1000 mg/kg bw/day at the necropsy. The spleen weights were significantly elevated in female animals at 1000 mg/kg bw/day at the termination of the treatment period. Minor differences with respect to the control were detected in female animals at 1000 mg/kg bw/day at the end of the recovery period. However there were no related histopathological alterations in the spleen tissue therefore spleen weight changes was considered to be reversible. Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 1000 mg/kg bw/day. Hyperplasia of spleen – in the red pulp (the erythroid, myeloid cells and the megakaryocytes) and in the white pulp (the peri-arteriolar lymphoid sheath, the number and size of lymphoid follicles and the size of marginal zones) – was detected in female animals at 1000 mg/kg bw/day at the termination of the treatment. Splenic hyperplasia was reversible as it was not present in high dose treated female animals at the end of the treatment period. Lymphocytic and histiocytic infiltration was observed in the liver, in some portal triads in one male animal in some female. This phenomenon could be in connection with the splenic hyperplasia and could be considered as the consequence of the possible stimulative impact of the 1000 mg/kg bw/day dose of test item as well.

Under the conditions of the present study, 1000 mg/kg bw/day dose of test item induced alterations in the spleen in the form of macroscopically enlargement associated with changes in some parameters of hematology (white blood cells, eosinophilic granulocytes and reticulocytes) or clinical chemistry (aspartate aminotransferase and total bilirubin) and increase in spleen weight accompanied by splenic hyperplasia (erythroid, myeloid and lymphoid) in female animals after consecutive 90-day oral (gavage) administration to Hsd.Han:Wistar rats. Splenic alterations were considered to be reversible although minor changes in the spleen weight were detected in female animals without any related histological alteration at the end of the recovery period.

There were no toxicologically relevant changes in the examined parameters in male or female animals at 300 mg/kg bw/day or at 100 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL: 1000 mg/kg bw/day for male Hsd.Han:Wistar rats

NOAEL: 300 mg/kg bw/day for female Hsd.Han:Wistar rats