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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
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:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
version of 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Product number M2933; Batch no. SLBN0344V
- Expiration date of the lot/batch: 30.04.2020
- Purity: 100.2% (Titr. by NaOH, anhyd.)
- Water content: 7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (RT)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle:soluble in vehicle up to the highest concentration (200 mg/mL). Test item proved to be stable in the vehicle at RT for at least 4 days (preparation period)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain: Hsd.Han (of Wistar origin)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., 1103 Budapest, Cserkesz u. 90
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks (both sexes)
- Weight at study initiation: males: 327-381g
females: 202-244g
- Fasting period before study: no
- Housing: before mating: 2 animals of the same sex/cage; mating: 1 male and 1 female/cage; pregnant females: individually; males after mating: 2/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY: food: ssniff SM R/M-Z+H complete diet, changed weekly; tap water, fresh every day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3°c
- Humidity (%): 30-70%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was solved in distilled water in concentrations of 20, 60 and 200 mg/mL. Dosing solutions were prepared not longer than for four days before the administration and stored at room temperature until use.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Batch no. of Aqua purificata (distilled water):
1702-5502, 1702-5510, 1703-5503 (supplier: Parma Produkt Kft., Budapest, Hungary)

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations was performed by LC/MS/MS in the Analytical Laboratory of Test Facility twice during the study. Recoveries of the test item in the dosing formulations ranged from 100 - 105% of the nominal concentrations.
Stability and homogeneity in distilled water over the range of relevant concentration has been demostrated at room temperature for at least four days (recoveries from 98% to 105%), i.e. the maximum period of solutions administered.
Duration of treatment / exposure:
56-66 days (depending on mating effectiveness)
Frequency of treatment:
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:
12 male and 12 female per dose and control
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results from a 14-day dose-range finding experiment that used the same concentrations
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): random
Positive control:
none

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (starting on Day 0)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes (body weight gain only)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: approx. 16 hours after last treatment (food deprived)
- Anaesthetic used for blood collection: Yes (isofluran)
- Animals fasted: Yes (approx. 16 hours)
- How many animals: 5 males and 5 females per group (randomly selected)
- Parameters examined:
- white blood cell (leukocyte) count (WBC)
- red blood cell (erythrocyte) count (RBC)
- hemoglobin concentration (HGB)
- hematocrit (HCT)
- Mean Corpuscular (erythrocyte) Volume (MCV)
- Mean Corpuscular (erythrocyte) Hemoglobin (MCH)
. Mean Corpuscular (erythrocyte) Hemoglobin Concentration (MCHC)
- platelet (thrombocyte) count (PLT)
- reticulocytes (RET)
- differential white blood cell count
- activated partial thromboplastin time (APTT)
- prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: approx. 16 hours after last treatment (food deprived)
- Animals fasted: Yes (approx. 16 hours)
- How many animals: 5 males and 5 females per group (randomly selected)
- Parameters examined:
- alanine aminotransferase activity (ALT)
- aspartate aminotransferase activity (AST)
- total bilirubin concentration (TBIL)
- creatinine concentration (CREA)
- Urea concentration (UREA)
- glucose concentration (GLUC)
- cholesterol concentration (CHOL)
- bile acids (BAC)
- sodium concentration (Na+)
- potassium concentration (K+)
- albumin concentration (ALB)
- total protein concentration (TPROT)
- T4 (in parental males at termination day 55)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional operation battery was performed on 5 animals per sex and group during the last exposure week but before blood sampling. Investigations included sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity. General physical conditions and behaviour of animals were tested (modified Irwin test).

IMMUNOLOGY: No

OTHER: none
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded (see reproductive toxicity study). The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, adrenal glands and pituitary and all organs showing macroscopic lesions of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution. Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.

All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides, see above) for five male and five female animals randomly selected from each group:
- adrenal glands
- bone with bone marrow and joint (femur)
- brain (representative regions: cerebrum, cerebellum, pons and medully oblongata)
- eyes (lachrymal gland with Harderian glands)
- female mammary gland
- gonads (testis with epididymides, ovaries, uterus with fallopian tube and vagina)
- gross lesions
- heart
- Kidneys
- large intestines (caecum, colon, rectum, incl. Peyer's patches)
- liver
- lungs (with main stem bronchi)
- lymph nodes (submandibular, mesenteric)
- muscle (quadriceps)
- esophagus
- pancreas
- pituitary
- prostate
- salivary glands (submandibular)
- sciatic nerve
- seminal vesicle with coagulating gland
- skin
- small intestines (representative regions: duodenum, ileum, jejunum)
- spinal cord (at three levels: cervical, mid-thoracic and lumbar)
- spleen
- sternum
- stomach
- thymus
- thyroid + parathyroid
- trachea
- urinary bladder

HISTOPATHOLOGY : Yes (control and high dose group)
Full histopathological examinations were performed on the following preserved organs and tissues of selected animals in the control and high dose groups (5 per sex):
- Adrenal glands
- Aorta
- Bone marrow
- Brain
- Cecum
- Colon
- Duodenum
- Eyes + optic nerves
- Esophagus
- Harderian glands
- Heart
- Ileum
- Jejunum
- Kidneys
- Lachrymal glands
- Liver
- Lungs
- Mesenteric lymph nodes
- Muscle (quadriceps)
- Pancreas
- Pituitary
- Prostate
- Rectum
- Salivary glands (subm.)
- Sciatic nerve
- Skin
- Spinal cord
- Spleen
- Sternum
- Stomach - forestomach
- Subm. lymph nodes
- Thymus
- Thyroid + parathyroid
- Trachea
- Urinary bladder

Histopathological examinations were performed on the following preserved organs and tissues of ALL animals in the control and high dose groups (12 per sex):
- Ovaries (Primodial, secondary and teriary follicles; Corpora lutea)
- Uterus
- Vagina
- Testes
- Epididymides
- Seminal vesicle with coagulating gland
- thymus (one male in low dose group due to hemorrhage
Other examinations:
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported.

In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
The thyroid weight after fixation was not determined in the absence of changes in thyroid hormones levels.
Statistics:
Depending on variance homogeneity between groups (Bartlett's test), parametric or non-parametric (Kruskal-Wallis test) ANOVA was performed, with subsequent inter-group comparisons (Duncan multiple range test or Mann-Whitney U-Test) in case of significant ANOVA results. If applicable, the Chi-square test was performed.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Daily clinical observation:
No treatment-related signs of systemic toxicity were detected at any dose level at the daily clinical observations (100, 300 and 1000 mg/kg bw/day, male or female).

Detailed weekly clinical observation:
The behavior and physical condition of animals was not affected by the test item at any dose level (100, 300 or 1000 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in any group during the course of study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item related effects were noted on the mean body weight and body weight gain values at 100, 300 or 1000 mg/kg bw/day. There were no significant differences between the control and test item treated groups in the body weight or body weight gain at any occasion.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no pathological changes in the examined hematological and blood coagulation parameters in any of the groups (male and female, 100, 300 or 1000 mg/kg bw/day).
The examined hematological and blood coagulation parameters were comparable in male and female animals of control and all test item treated groups and statistical significance was not observed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).
Some sporadic statistically differences in some parameters were not related to dose and considered of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observations did not demonstrate any test item related changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination are considered to be normal in all groups (100, 300 and 1000 mg/kg bw/day, control).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse effects on the examined organ weights in male or female animals at 100, 300 or 1000 mg/kg bw/day.
Kidney weights (absolute and relative) were reduced in a single male rat in the mid dose group. This minor change was not related to doses, the values met the historical controls and no corresponding histopathological correlate was observed. Therefore, this is considered to have no toxicological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some findings were only present in single animals (of both sexes) in most groups including control. In the absence of corresponding histopathological findings, these were considered to be not toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of male or female animals at 1000 mg/kg bw/day. No test item related alterations in organs and tissues and no morphological evidence of acute or subacute injury to the various organs were observed.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No test item related alterations in organs and tissues an no morphological evidence of acute or subacute injury to the various organs were observed.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone levels:
There were no significant differences with respect to the control in the T4 thyroid hormone levels in parental male animals or in offspring sampled on postnatal day 13 at any dose levels.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

The systemic toxicity of the test substance was investigated in an experimental study according to OECD test guideline 422 under GLP-conditions. The substance, solved in distilled water, was administered to Wistar rats of both sexes by oral gavage in doses of 100, 300 and 1000 mg/kg bw daily for 56 to 66 days (depending on mating effectiveness). Observing mortality, clinical signs, body weight, body weight gain, food consumption, hematology, blood coagulation and clinical chemistry parameters, macroscopic alterations, organ weights and histopathological alterations, no treatment-related toxicological effects were found. Therefore, the substance was considered to be not systemically toxic in this repeat dose toxicity study.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, the test item did not cause signs of systemic toxicity in parental male and female Hsd.Han: Wistar rats at 100, 300 or 1000 mg/kg bw/day doses administered by oral gavage.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

Executive summary:

The systemic toxicity of the test substance was investigated in a experimental study according to OECD test guideline 422 under GLP-conditions. The substance, solved in distilled water, was administered to Wistar rats of both sexes by oral gavage in doses of 100, 300 and 1000 mg/kg bw daily for 56 to 66 days (depending on mating effectiveness). Observing mortality, clinical signs, body weight, body weight gain, food consumption, hematology, blood coagulation and clinical chemistry parameters, macroscopic alterations, organ weights and histopathological alterations, no treatment-related toxicological effects were found. Therefore, the substance was considered to be not systemically toxic in this study.