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Toxicological information

Repeated dose toxicity: oral

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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:
2020

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate cages
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females housed individually.
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
vegetable oil
Remarks:
sunflower oil
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males dosed for at least 28 days (14 days pre-mating and 14 days mating plus an optional extended post-mating period until the necessary number of pregnant female animals is evident)
Females dosed for 14 days pre-mating, through 14 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice.
Frequency of treatment:
daily
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
Observations on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention will be directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted on five male and five female animals randomly selected from each group during their respective last exposure week but before the blood sampling.

BODY WEIGHT: Yes
- Time schedule for examinations:
Parental males were weighed on the first day of dosing (day 0), at least weekly thereafter and at termination.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally measured on gestation days 10 in order to give accurate treatment volumes.

FOOD CONSUMPTION: Yes
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase as follows: premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals. Fasted body weight was determined for animals selected for toxicity examinations before the necropsy.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Anaesthetic used for blood collection: Yes, Isofluran anesthesia
- Animals fasted: Yes
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked: WBC (White Blood Cell leukocyte count), RBC (Red Blood Cell erythrocyte count), HGB (Hemoglobin concentration), HCT (Hematocrit, relative volume of erythrocytes), MCV (Mean Corpuscular erythrocyte Volume), MCH (Mean Corpuscular erythrocyte Hemoglobin), MCHC (Mean Corpuscular erythrocyte Hemoglobin Concentration), PLT (Platelet thrombocyte count), RET (Reticulocytes), Differential white blood cell count, APTT (Activated partial Thromboplastin Time), PT (Prothrombin Time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Animals fasted: Yes, Isofluran anesthesia
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked: ALT (Alanine Aminotransferase activity), AST (Aspartate Aminotransferase activity), TBIL (Total Bilirubin concentration), CREA (Creatinine concentration), UREA (Urea concentration), GLUC (Glucose concentration),CHOL (Cholesterol concentration), Na+ (Sodium concentration),K+ (Potassium concentration), ALB (Albumin concentration), TPROT (Total Protein concentration)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (T4,TSH) as follows:
- from all dams and at least two pups per litter on day 13 if feasible
- from all parent male animals at termination
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The appearance of the tissues and organs were observed, and any abnormality was recorded including details of the location, color, shape and size.
At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals was determined. In addition, for five males and females randomly selected from each group, adrenal glands,brain, heart, kidneys, liver, spleen and thymus was weighed. Absolute organ weight was reported. Relative organ weight (to body and brain weights) was calculated and reported.
The thyroid weight was determined after fixation.

HISTOPATHOLOGY: Yes
Detailed histological examination were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments ofthe ovary as well as the epithelial capsule and ovarian stroma.
In addition, these organs were processed and examined histologically in not mated, non-pregnant or not delivered females and males these females cohabited with in the low and middle dose groups.
Full histopathology examinations was performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. Histopathological examinations were performed for the pancreas in all dose groups.
Other examinations:
LITTER OBSERVATIONS:
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) presence of gross anomalies. Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete, post-natal day 0) and on postnatal day 4. The anogenital distance of each pup was determined on postnatal day 4. Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum T4 and TSH levels. The number of nipples/areolae in male pups was counted on postnatal day 13.

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).
Statistics:
The homogeneity 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. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.

Results and discussion

Effect levels

Key result
Dose descriptor:
NOAEL
Sex:
male/female
Remarks on result:
other: study on-going, results will be presented in an update

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion