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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:
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:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories GmbH, Sulzfeld, Germany
- Age at study initiation: 34 ± 1 days
- Housing: polysulfonate cages TECNIPLAST, Hohenpeißenberg, Germany
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”, meal, Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): yes

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Test substance solution was prepared with drinking water to the desired volume, subsequently mixed by a magnetic stirrer. The test-substance preparations were produced at least weekly and stored at room temperature. The administration volume was 10 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance in drinking water at room temperature for a period of 7 days was demonstrated during the administration period.
Homogeneity was verified in 3 samples in the highest and lowest concentration at the beginning of the studyand towards the end of the administration period.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
360 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
40 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:
- Time schedule: twice daily on working days and once daily on Saturdays, Sundays and public holidays

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals

BODY WEIGHT:
- Time schedule for examinations: before the start of the administration period. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight
on day 0 was calculated as body weight change.

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: Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Prior to the start of the administration period on day -1 the eyes of all animals and on study day 91 the eyes of the control and high-dose animals.

HAEMATOLOGY:
- Time schedule for collection of blood: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.


CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS:
individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery (FOB) was performed in all animals at the end of the administration period.
Motor activity (MA) was also measured.

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Vaginal smears for estrous cycle determination were prepared in the morning and evaluated according to the timetable for at least 3 weeks.
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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Anesthetized animals, Adrenal glands, Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix,

HISTOPATHOLOGY: Yes
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix,Coagulating gland, Colon, Duodenum, Epididymis, left, Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Mammary gland (male and female), Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testis, left, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related, adverse effects were observed in male and female animals.
All male and female animals of test group 3 (360 mg/kg bw/d) showed salivation within 2 hours after administration on several days of the administration period, starting from study day 23 onwards. In addition, one female animal of test group 2 (120 mg/kg bw/d) showed salivation within 2 hours after administration from study day 48 to study day 51. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract.
Mortality:
no mortality observed
Description (incidence):
One male animal of test group 3 (360 mg/kg bw/d) died accidentally during blood sampling on study day 92 shortly before necropsy.
One female animal of test group 0 (0 mg/kg bw/d) was sacrificed in a moribund condition on study day 71.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights were significantly lower in male animals of test group 3 (360 mg/kg bw/d) between study days 63 to 91 with a maximum of -10.8% on study day 91. In addition, mean body weight change values were significantly lower in male animals of test group 3 (360 mg/kg bw/d) between study days 49 to 91 with a maximum of -18.1% on study days 63 and 91. These changes were assessed to be treatment-related and adverse.
In females animals of test group 3 (360 mg/kg bw/d) mean body weights were only decreased on individual study days, i.e. study days 28 (-5.6%) and 56 (-5.3%). The same was true for mean body weight change values, which were only lower on study days 28 (-11.7%) and 56 (-9.1%). As these changes occurred only temporarily during the course of the administration period and body weight parameters were not influenced at the end of the study, the changes were assessed to be incidental and not related to treatment.
No significant changes in body weight parameters were observed for male and female animals of test groups 1 and 2 (40 and 120 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, absolute neutrophil and eosinophil counts as well as relative eosinophil counts were increased in male animals of test group 3 (360 mg/kg bw/d). In males of test group 2 (120 mg/kg bw/d), absolute and relative eosinophil cell counts were already higher compared to controls. However, this was the only changed hematology parameter in these individuals and, therefore, the alteration was regarded as treatment related but not adverse (ECETOC Technical Report No. 85, 2002).
In male and female animals of test group 3 (360 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was prolonged. Prothrombin time was already higher in females
of test group 2 (120 mg/kg bw/d) compared to controls, but in this group the values were within the historical control range (HQT 31.1-36.8 sec) and, therefore, the prothrombin
alteration was regarded as incidental and not treatment-related for animals of test group 2.
Some other hematology parameters in various test groups were changed but the values were within historical control ranges and regarded as incidental and not treatment-related:
Male animals: decreased mean corpuscular hemoglobin content (MCH) in test group 3 (360 mg/kg bw/d; MCH 1.00-1.10 fmol); increased platelet counts in test groups 1, 2 and 3 (40, 120 and 360 mg/kg bw/d; platelets 643-875 Giga/L);
Female animals: increased total white blood cell (WBC) counts in test groups 2 and 3 (WBC 2.61-4.15 Giga/L); increased absolute and relative lymphocyte counts in test groups 1, 2 and 3 (absolute lymphocytes 2.05-3.35 Giga/L; relative lymphocytes 69.2-82.4%).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, in rats of both sexes of test group 3 (360 mg/kg bw/d) total bilirubin values were increased. In males of test group 3 the higher bilirubin
mean was within the historical control range (total bilirubin 0.56-2.76 μmol/L). In females of test group 2 (120 mg/kg bw/d), total bilirubin values were already higher compared to
controls, but the values were also within the historical control range (total bilirubin 1.07-3.12 μmol/L). Therefore, the alterations in males of test group 3 as well as in females of test
group 2 were regarded as incidental and not treatment-related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.

Motor activity measurement
Regarding the overall motor activity as well as the single intervals, no test substance related deviations were noted.
Comparing the single intervals with the control groups, significantly decreased values were measured at interval 5 for male animals of test groups 1 and 3 (40 and 360 mg/kg bw/d) as well as at interval 7 for male animals of test groups 2 and 3 (120 and 360 mg/kg bw/d). In female animals of test group 3 (360 mg/kg bw/d) the single value of interval 4 was significantly increased.
As only single intervals showed deviating values and overall motor activity did not reveal significant changes in test groups 1-3 (40, 120 and 360 mg/kg bw/d), the findings were
regarded as being incidental and not related to treatment.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute organ weights:
When compared to control group 0 (set to 100%), the mean absolute weight of the liver of female test group 3 animals was significantly increased. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights:
See table 1 for mean relative weights compared to control group 0 (set to 100%). All other mean relative weight parameters did not show significant differences when
compared to the control group 0.
The increased relative liver weights in male animals of test group 2 and 3 (120 and 360 mg/kg bw/d) correlated with hepatocellular hypertrophy and were therefore assessed as
treatment-related.
In females, both absolute and relative liver weights of test group 3 were increased and correlated with hepatocellular hypertrophy. Therefore, the increased relative liver weights in males of test groups 2 and 3 and the increased absolute and relative liver weights in females of test group 3 were regarded as treatment-related.
The increase of relative weights of thyroid glands of male animals of test groups 2 and 3 was regarded as incidental as the weights were within historical controls of both absolute and relative weights and thyroid glands did not show a histopathological finding.
The terminal body weights in all treated males were not statistically significantly decreased but laid below historical controls (in this study: test group 1: 334.27 g; test group 2: 341.43 g; test group 3: 322.811 g; historical controls minimum 352.84 g) causing an increase in relative weights in males in heart (all treated test groups), testes (test groups 2 and 3) and kidney (test groups 1 and 3).
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hepatocellular hypertrophy was observed in the liver of male and female animals of test groups 2 and 3. The hypertrophy was centrilobular and characterized by not only an
enlargement of the cytoplasm but also with higher severity grades (moderate, severe) an enlargement of the nuclei (see table 2).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test substance-related effects on estrous cycle length and the number of cycles were obtained.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
360 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 1: Relative organ weights

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(40)

2

(120)

3

(360)

1

(40)

2

(120)

3

(360)

Heart

113%**

109%*

114%*

 

 

 

Kidneys

108%*

103%

108%*

 

 

 

Liver

107%

108%**

119%**

99%

103%

118%**

Testes

104%

111%*

112%**

 

 

 

Thyroid glands

110%

122%**

118%*

 

 

 

Table 2: Histopathological findings

 

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(40)

2

(120)

3

(360)

0

(0)

1

(40)

2

(120)

3

(360)

No. of animals

10

10

10

10

10

10

10

10

Hypertrophy, centrilobular

0

0

10

10

0

0

7

10

  • Grade 1

 

 

10

 

 

 

6

 

  • Grade 2

 

 

 

6

 

 

1

1

  • Grade 3

 

 

 

2

 

 

 

7

  • Grade 4

 

 

 

2

 

 

 

2

Applicant's summary and conclusion

Conclusions:
The test substance was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; drinking water served as vehicle control, 40 mg/kg bw/d, 120 mg/kg bw/d and 360 mg/kg bw/d over a period of 3 months. In addition to the required examinations, special attention was given to the reproductive organs of male and female animals.
Food consumption and body weights were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. In addition, the animals were examined daily for any clinically abnormal signs. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. For at least 3 weeks an estrous cycle determination was performed. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period.
Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations. Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals for sperm examinations.


As a result, analytical examinations confirmed the stability of the test-substance preparations for a period of 7 days at room temperature, the homogeneous distribution of the test substance in the vehicle and the correctness of the prepared concentrations.

At 360 mg/kg bw/d clinical examinations have shown that mean body weights were significantly decreased in male animals between study days 63 to 91 with a maximum of -10.8% on study day 91. Mean body weight change values were significantly reduced in male animals between study days 49 to 91 with a maximum of -18.1% on study days 63 and 91.
Clinical Pathology rrevealed increased total bilirubin values in female animals, prolonged prothrombin time in both sexes, increased absolute neutrophil and absolute and relative eosinophil cell counts in males and increased γ-glutamyl transferase (GGT) activities in females.
Pathology has shown centrilobular hypertrophy in all males and females graded up to severe in some animals of both sexes with markedly enlarged nuclei in association with increased liver weights (males: relative 119%; females: absolute 113%, relative 118%)

At 120 mg/kg bw/d and 40 mg/kg bw/d clinical examinations, clinical pathology and pathology did not show treatment-related, adverse effects.



Executive summary:

In conclusion the administration of the test substance by gavage to male and female Wistar rats for 3 months caused test substance-related adverse signs of systemic toxicity at a dose level of 360 mg/kg bw/d in male and female Wistar rats.

Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 120 mg/kg bw/d in male and in female Wistar rats.