Registration Dossier

Administrative data

Endpoint:
short-term repeated dose 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:
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:
yes
Remarks:
minor, without any effect on study results and validity
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

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: 13 weeks (both sexes)
- Weight at study initiation: males: 359-434g
females: 209-250g
- 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: 27 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 substance was solved in distilled water in concentrations of 20, 60 and 200 mg/L

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: 20, 60 and 200 mg/L
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Batch no. (if required): 1610-5527; 1701-5524; 1702-5502 (supplied by Parma Produkt Kft., Budapest, Hungary)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity in distilled water over the range of relevant concentration has been demostrated at room temperature for at least five days (approx. 100%), i.e. the maximum age of solutions administered.
Duration of treatment / exposure:
55-67 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 findings 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
- Cage side observations: not further specified

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: appro., 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)
- 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)
- glucose concentration (GLUC)
- cholesterol concentration (CHOL)
- total bilirubin concentration (TBIL)
- 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: No

IMMUNOLOGY: No

OTHER: none
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- adrenal glands
- bone with bone marrow and joint (femur)
- brain (representative regions: cerebrum, cerebellum, pons and medulla oblongata)
- eyes (lachrymal gland with Harderian glands)
- female mammary gland
- gonads (testis with epididymides, ovaries, uterus with fallopian tube and vagina)
- gross lesions
- heart
- large intestines (caecum, colon, rectum, incl. Peyer's patches)
- liver
- lungs (with main stem bronchi)
- 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 lumber
- spleen
- sternum
- stomach
- thymus
- thyroid + parathyroid
- trachea
- urinary bladder

HISTOPATHOLOGY : Yes (control and high dose group)
- ovaries
- uterus
- vagina
- testes
- epididymides
- prostate
- seminal vesicle with coagulating gland
- thymus (one male inlow dose group due to hemorrhage
Other examinations:
organ weights:
- brain
- testes
- epididymides
- prostate
- seminal vesicle with coagulating gland
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):
In the 300 mg/kg bw dose group, brownish fur around the right eye of one male rat and alopecia on the chest of one female rat were observed. These findings occur sporadically in experimental rats and were not related to the treatment.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
effects observed, non-treatment-related
Description (incidence and severity):
Some sporadic statistically differences in some parameters (lower mean percentage of monocytes and basophil granulocytes, and a higher mean percentage of reticulocytes at 100 mg/kg bw/day; higher mean percentage of reticulocytes at 300 mg/kg bw/day; lower mean percentage of neutrophil granulocytes and a higher mean percentage of lymphocytes at 1000 mg/kg bw/day) were not related to dose and considered of no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some sporadic statistically differences in some parameters (higher mean sodium concentration at 100 mg/kg bw/day; elevated urea concentrations at 300 mg/kg bw/day (females) and elevated glucose concentrations at all doses (female)) were not related to dose and considered of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Some sporadic statistically differences in some parameters (reduced mean weights of adrenal glands (absolute and relative organ weights) in males at all treatment doses; lower brain and heart weights relative to body weight at 300 mg/kg bw/day (males)) were not related to dose and considered of no toxicological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Findings were only present in single rats of both sexes in all groups including control, such as pale and smaller seminal vesicle (1 male of control), thymic hemorrhage (one male at 100 mg/kg bw/day and one female at 1000 mg/kg bw/day), slight hydrometra (1 female at 100 mg/kg bw/day, 2 females at 300 mg/kg bw/day, 1 female at 1000 mg/kg bw/day). In the absence of corresponding histopathological findings and a dose relation, these were considered to be not toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of male and female rat at 1000 mg/kg bw/day (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulation gland in all animals; full histopathology in selected animals).
In one control male a decreased amount of secretum in the seminal vesicle (one side) was observed, which was considered as an individual disorder without toxicological significance.
In the other male rats, the investigated organs of the reproductive system were histologically normal. The various spermatogenic cells were the same in quantity and morphologically in the testes of controls and treated groups. Histology of the epididymides, seminal vesicles and coagulating glands was normal in all animals.
In the female rat the investigated organs of the reproductive system were histologically normal. The cortical region of the ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating active maturation of oocytes, and ovulation Also the epithelial capsule and ovarian stroma was normal in all cases.
Some lesions were noted (dilatation of uterine horns of one female at 1000 mg/kg bw/day; pulmonary alveolar emphysema in two males and 2 females of the control and two males at 1000 mg/kg bw/day; alveolar histicytosis in one control male; hyperplasia of bronchus associated lymphoid tissue in one control female and two females at 1000 mg/kg bw/day; acute hemorrhage in the thymus in one male at 100 mg/kg bw/day and one female at 1000 mg/kg bw/day) .
Pulmonary emphysema and acute hemorrhage were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Alveolar histicytosis is a common incidental findings in elder rat. Hyperplasia of bronchus associated lymphoid tissue is a physiological immune-morphological phenomenon without toxicological significance. Dilatation of the uterine horns is indicative of a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (pro-estrous phase) of uterus without pathological significance.
Histopathological findings: neoplastic:
no effects observed

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 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 55 to 67 days. 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.

Applicant's summary and conclusion

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 55 to 67 days. 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.