Registration Dossier

Administrative data

Description of key information

In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422, required in Annex VIII, Section 8.6.1 of REACH regulation, the test item did not cause signs of systemic toxicity in parental male and female Han: WIST rats at 100, 300 or 1000 mg/kg bw/day doses administered by oral gavage, either.

A NOAEL of 1000 mg/kg bw/day was determined.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January 2018 - 24 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity w ith the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Toxi-Coop ZRT, 8230 Balatonfüred, Arácsi út 97, Hungary
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: VPS9016001
- Expiration date of the lot/batch: 27.06.2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (15-25°C)
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 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: Young adult rats, approximately 13 weeks old at the beginning of the pre-mating phase and 15 weeks at mating.
- Weight at study initiation: The weight variation in animals involved at the starting point of the study not exceed ± 20 % of the mean group weight of each sex.
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females will be housed individually.
Males after mating: 2 animals/cage
- 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 from municipal supply
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY:
The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The drinking water is periodically analyzed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. The pH of the formulations was adjusted to pH=4-5 with 10 N NaOH. Formulations were prepared not longer than for three days before the use.

VEHICLE
- Concentration in vehicle: 20 mg/mL, 60 mg/mL and 200 mg/mL
- Treatment volume: 5 mL dose preparation/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and at least three replicates are measured on 2 occasions, approximately during the first and last treatment weeks. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.
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
Dose / conc.:
0 mg/kg bw/day (nominal)
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:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose setting is based on findings obtained in a 14-day dose range finding study performed with the substance and in agreement with the Sponsor. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. In case of severe signs of toxicity, the high dose will be reduced.
Positive control:
None
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
Parental males weighed on the first day of dosing (day 0), weekly thereafter and at termination. Parental females 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 additionally measured on gestation day 10 in order to give accurate treatment volumes, but these data was evaluated statistically. Body weight data was reported individually for adult animals. Body weight was measured on the day of necropsy for animals subjected to organ weighing (all male animals and females selected for further examinations).

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

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.

HISTOPATHOLOGY: Yes
Detailed histological examination was 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 of the ovary as well as the epithelial capsule and ovarian stroma.
The following organs were preserved: Adrenal glands, Aorta, Bone with marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Eyes (lachrymal gland with Harderian glands), Female mammary gland, Gonads (testes with epididymides, ovaries, uterus with vagina), Gross lesions, Heart, Kidneys, Large intestines (caecum, colon, rectum, including Peyer’s patches), Liver, Lungs (with main stem bronchi; inflation with fixative and then
immersion), 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.

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.

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals (n=5 animals/sex/group) in the control and high 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). Dead pups found were subjected to necropsy by a macroscopic examination. Any observed abnormalities were recorded.
Statistics:
The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0. 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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Soft stool and nuzzling up the bedding material were noted for all male and female animals at 1000 mg/kg bw/day. The nuzzling up the bedding material was with short duration after the treatment and there were no soft stool related body weight changes therefore these signs were considered to be toxicologically not relevant. There were no clinical signs in animals in the control (12/12 male and 12/12 female), 100 mg/kg bw/day (12/12 male and 11/12 female) and 300 mg/kg bw/day (12/12 male and 12/12 female). Slight alopecia on the thorax of one female rat (~1cm in diameter) was observed at 100 mg/kg bw/day (1/11) between gestation days 17 and 21. This clinical sign was considered to be individual finding and not related to the treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
There was no mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight development was not adversely affected by the test item in male or female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period. Statistical significance with respect to the control was detected in female animals at 100, 300 and 1000 mg/kg bw/day at the higher mean body weight gain between gestation days 14 and 21 and in the whole gestation period (gestation days 0-21). The mean body weight was also significantly higher than in the control group at 1000 mg/kg bw/day on gestation day 21. These changes in the body weight and body weight gain were considered to be related to the lower mean number of fetuses in the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item or treatment related adverse changes in the food consumption of male and female animals at any dose level (100, 300 and 1000 mg/kg bw/day). Statistical significance was noted for the slightly higher mean daily food consumption in female animals at 300 and 1000 mg/kg bw/day between gestation days 14 and 21. The increased food consumption was in full compliance with the increased body weight gain and higher number of fetuses and was not judged to be of toxicological relevance.
Food efficiency:
not examined
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):
In male animals, statistical significances were detected at the lower mean percentage of monocytes (MONO) and at the higher mean platelet (thrombocyte) count (PLT) at 100 mg/kg bw/day when compared to their control. In the lack of any dose relationship, these findings were considered to have no toxicological relevance.
Slightly but statistically significantly higher mean hemoglobin concentration (HGB) and hematocrit (relative volume of erythrocytes) (HCT) were detected in male animals at 300 and 1000 mg/kg bw/day. All the individual values of these two parameters met well the historical control. Moreover, the control values of HGB are slightly lower than the historical control mean. Therefore, these slight differences in HGB and HCT were judged to have no biological significance. In female animals, statistical significances were detected at the slightly lower mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) and at the higher mean percentage of monocytes (MONO) at 300 mg/kg bw/day when compared to their control. Similar findings were not seen at the high dose treated animals these were considered to have no toxicological relevance.
Statistically significantly lower mean red blood cell (erythrocyte) count (RBC) was observed in female animals at 1000 mg/kg bw/day. In the lack of related changes this finding was judged to have no toxicological meaning.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, statistical significance was detected with respect to their control at the lower mean activity of aspartate aminotransferase (AST) and at the higher mean urea concentration (UREA) at 100 mg/kg bw/day. At 300 mg/kg bw/day, the mean sodium (Na+) concentration was slightly higher than in their male control group. In male animals at 1000 mg/kg bw/day, higher mean alanine aminotransferase activity (ALT), higher mean concentrations of urea and mean total bilirubin (TBIL) and lower mean potassium concentration (K+) concentration were observed when compared to their control. The mentioned differences between control and test item treated male animals reached statistical significances however these slight changes were judged to have no toxicological relevance. The slightly elevated ALT concentration is in good correlation with the increased mean liver weight of male animals in high dose group. In the lack of any histopathological findings this change was considered to be an adaptive one.
Other statistical differences were with minor degree (AST, UREA, Na+, K+ and TBIL) or not related to doses therefore these findings were considered to be of low or no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Slight changes in the liver and kidney weights with respect to their controls might be related to a test item influence on hepatic or renal function in male animals at 300 or 1000 mg/kg bw/day taking into account the changes in urea or ALT levels. However the degree of all of these changes was small and there were no supporting histopathological findings. Therefore these findings were considered to be indication of the adaptation process and the toxicological significance is equivocal. Changes in thymus weights were not dose related (were only detected in the low dose treated male animals) therefore were without toxicological relevance. Statistical significances noted for some other organs (liver, kidneys and heart, each relative to brain weight) and fasted body weight relative to brain weight in female animals were with small degree and were not associated with any clinical pathological or histopathological findings therefore were considered to be toxicologically not relevant. The slightly increased spleen weights with respect to the control in female animals at 1000 mg/kg bw/day were not accompanied by clinical pathology or histopathological lesions therefore were considered to be of little or no biological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg bw/day and thymic hemorrhage was observed in one male animal (1/12).
At 300 mg/kg bw/day, pea-sized formation was detected at the cardia of the stomach in one male animal (1/12).
The organs and tissues were judged to be normal of male animals at 1000 mg/kg bw/day group (12/12) during the course of necropsy.
Hydrometra was detected in two dams in the control group (2/8) at the necropsy.
There were no macroscopic changes in the organs or tissues in the other dams in control (6/8), at 100 mg/kg bw/day (11/11), at 300 mg/kg bw/day (9/9) or at 1000 mg/kg bw/day (10/10) groups. In non-pregnant females, hydrometra (2/4 control, 1/2 at 300 mg/kg bw/day) and cyst on the left ovary (1/2 at 1000 mg/kg bw/day) was seen at the necropsy. The organs and tissues were judged to be normal in other non-pregnant female animals as follows: 2/4 control, 1/1 at 100 mg/kg bw/day, 1/2 at 300 mg/kg bw/day, 1/2 at 1000 mg/kg bw/day. Hydrometra was observed in the single not delivered female animal (1/1 at 300 mg/kg bw/day). Hemorrhage in the thymus was probably due to circulatory disturbance developed during exsanguination procedure. Pea-sized formation at the cardia in the stomach was considered to be an individual lesion in single male animal at 100 mg/kg bw/day supported by histopathological evaluation. It was probably a developmental disorder. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or other pathological lesion) these were judged not to be toxicologically relevant. Cyst in the ovary in a single non-pregnant female at 1000 mg/kg bw/day was considered to be individual change. This lesion is common in not treated experimental rats of this strain.
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 or female animals at 1000 mg/kg/bw/day (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals; full histopathology in selected animals). In all male animals, the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism in all cases. In some female animals (1/1 not delivered and 1/2 non-pregnant at 300 mg/kg bw/day) dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
In selected animals, alveolar emphysema in minimal degree was observed in the lungs in the control (2/5 dams) and at 1000 mg/kg bw/day (1/5 male, 1/5 dam). This finding was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Hyperplasia of bronchus associated lymphoid tissue (BALT) was noted in the control group (2/5 male, 1/5 dam) and at 1000 mg/kg bw/day (2/5 male, 1/5 dam). Hyperplasia of BALT is a physiological immune-morphological phenomenon, without toxicological significance. Acute hemorrhage was observed in the thymus in one male at 300 mg/kg bw/day (1/1), which was processed histologically. This finding was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Focal dilatation in the non-glandular region of the stomach was observed in one male at 100 mg/kg bw/day (1/1) without degenerative or inflammatory lesions. This finding was considered as an individual phenomenon, probably a developmental disorder. There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the selected animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Serum Levels of Thyroid Hormones:
There were no statistically significant differences with respect to the control in the thyroid hormone (free T4 and TSH) levels in parental male animals.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse sysemic effects at highest dose tested
Key result
Critical effects observed:
no

Table 1: Summary of clinical observations males  

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Normal

12/12

12/12

12/12

0/12

Soft stool

0/12

0/12

0/12

12/12

Nuzzling up the bedding material

0/12

0/12

0/12

12/12

Table 2: Summary of clinical observations females

Pre-mating and mating periods

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Normal

12/12

12/12

12/12

0/12

Soft stool

0/12

0/12

0/12

12/12

Nuzzling up the bedding material

0/12

0/12

0/12

4/12

Post mating period

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Normal

4/4

1/1

2/2

0/2

Soft stool

0/4

0/1

0/2

2/2

Nuzzling up the bedding material

0/4

0/1

0/2

1/2

Gestation period

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Normal

8/8

10/11

10/10

0/10

Alopecia

0/8

1/11

0/10

0/10

Soft stool

0/8

0/11

0/10

10/10

Nuzzling up the bedding material

0/8

0/11

0/10

10/10

Lactation period

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Normal

8/8

11/11

9/9

0/10

Soft stool

0/8

0/11

0/9

10/10

Remark: Frequency of observations: number of animals (cage) with observation/number of animals (cage) examined

 

Table 3: Summary of necropsy findings (male)

Organs

Observations

Frequency of observations per group

 

Control

100

mg/kg bw/day

300

mg/kg bw/day

1000

mg/kg bw/day

No macroscopic findings

12/12

11/12

11/12

12/12

Stomach

Pea sized formation at the cardia

0/12

1/12

0/12

0/12

Thymus

Hemorrhage

0/12

0/12

1/12

0/12

 

Table 4: Summary of necropsy findings (female)

 

Organs

Observations

Frequency of observations per group

 

Control

100

mg/kg bw/day

300

mg/kg bw/day

1000

mg/kg bw/day

Dams

No macroscopic findings

6/8

11/11

9/9

10/10

Uterus

Hydrometra

2/8

0/11

0/9

10/10

Non-pregnat females

No macroscopic findings

2/4

1/1

1/2

1/2

Uterus

Hydrometra

2/4

0/1

1/2

0/2

Ovary

Cyst, left side

0/4

0/1

0/2

1/2

Not delivered females

Uterus

Hydrometra

-

-

1/1

-

Conclusions:
Based on the results the NOAEL for systemic toxicity of male/female rats was 1000 mg/kg bw/day.
Executive summary:

In this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test the test material was repeatedly administered orally (by gavage) from conception to day 13 post-partum to parental animals. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally once a day at 0 (vehicle), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, distilled water. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 50 days). Females were additionally exposed through the gestation period and up to lactation days 13-18 (altogether for 50-65 days). Besides the effects regarding reproduction/development (please refer ti IUCLID section 7.8) the observations included mortality, clinical signs, body weight and food consumption. Furthermore blood samples were collected for possible determination of serum levels of thyroid hormones (T4, TSH) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination.All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) in the control and high dose groups and in non-pregnant or not delivered females and the males these females cohabited with in the low and mid dose groups. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups. In addition, stomach for one male at 100 mg/kg bw/day and thymus for one male at 300 mg/kg bw/day were also processed and examined due to the necropsy observations (pea sized formation at the cardia; hemorrhage, respectively).The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (distilled water) only. Historical control data were also considered.There was no mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study.Adverse clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily or detailed weekly clinical observations or at the terminal functional observations. 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) during the entire observation period. However, test item treatment related soft stool was detected at 1000 mg/kg bw/day in male and female animals. Nuzzling up the bedding material was also observed at this dose in males and several females for a short period of time. Test item related adverse changes in the body weight or body weight gain were not detected. The body weight development was not disturbed and it was comparable in the control and test item treated groups. The mean daily food consumption was not affected by the test item in male or female animals at 100, 300 and 1000 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods). There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day. Specific alterations related to test item effect were not detected in the examined clinical chemistry parameters at any dose level (male or female; 100, 300 or 1000 mg/kg bw/day). There were no test item related changes in the serum thyroid hormone (T4, TSH) levels at any dose (parental male animals or PN13 offspring).Macroscopic alterations related to the effect of the test item were not found in male or female animals at 100, 300 or 1000 mg/kg bw/day at the necropsy. There were no test item related adverse changes in the weights (absolute and relative to body or brain weights) of selected organs in the animals at any dose level. Histopathological examinations of the investigated organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day. There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or in the 1000 mg/kg bw/day group.

The NOAEL for systemic toxicity of male/female rats was therefore considered to be 1000 mg/kg bw/day. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD TG 422 DRF

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Additional information

Key study:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test the test material was repeatedly administered orally (by gavage) from conception to day 13 post-partum to parental animals. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally once a day at 0 (vehicle), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, distilled water. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 50 days). Females were additionally exposed through the gestation period and up to lactation days 13-18 (altogether for 50-65 days). Besides the effects regarding reproduction/development (please refer ti IUCLID section 7.8) the observations included mortality, clinical signs, body weight and food consumption. Furthermore blood samples were collected for possible determination of serum levels of thyroid hormones (T4, TSH) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) in the control and high dose groups and in non-pregnant or not delivered females and the males these females cohabited with in the low and mid dose groups. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups. In addition, stomach for one male at 100 mg/kg bw/day and thymus for one male at 300 mg/kg bw/day were also processed and examined due to the necropsy observations (pea sized formation at the cardia; hemorrhage, respectively).The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (distilled water) only. Historical control data were also considered. There was no mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study. Adverse clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily or detailed weekly clinical observations or at the terminal functional observations. 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) during the entire observation period. However, test item treatment related soft stool was detected at 1000 mg/kg bw/day in male and female animals. Nuzzling up the bedding material was also observed at this dose in males and several females for a short period of time. Test item related adverse changes in the body weight or body weight gain were not detected. The body weight development was not disturbed and it was comparable in the control and test item treated groups. The mean daily food consumption was not affected by the test item in male or female animals at 100, 300 and 1000 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods). There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day. Specific alterations related to test item effect were not detected in the examined clinical chemistry parameters at any dose level (male or female; 100, 300 or 1000 mg/kg bw/day). There were no test item related changes in the serum thyroid hormone (T4, TSH) levels at any dose (parental male animals or PN13 offspring). Macroscopic alterations related to the effect of the test item were not found in male or female animals at 100, 300 or 1000 mg/kg bw/day at the necropsy. There were no test item related adverse changes in the weights (absolute and relative to body or brain weights) of selected organs in the animals at any dose level. Histopathological examinations of the investigated organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day. There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or in the 1000 mg/kg bw/day group.

The NOAEL for systemic toxicity of male/female rats was therefore considered to be 1000 mg/kg bw/day. 

Supporting study:

A study according OECD TG 407 was conducted to obtain initial information on the toxic potential of the test item after subacute repeated application in rats at three dose levels and to allow dose-setting for a subsequent Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test.

The test item was administered orally (by gavage) to Hsd.Han: Wistar rats (n=5 animals/sex/group) once a day at 0 (vehicle control), 100 300 and 1000 mg/kg bw/day corresponding to concentrations of 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw for 14 days. Analytical control of dosing formulations was performed once during the study.

The suitability of the chosen vehicle for the test item was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation.

Measured concentrations of formulations applied in the study varied in the acceptable range (between 92 % and 107 % of the nominal concentrations) and all formulations were homogenous, thereby confirming proper dosing.

Detailed clinical observations were made on all animals once a day, after treatment at approximately the same time. Body weight and food consumption were measured weekly. Clinical pathology and gross pathology examinations were conducted at the end of the treatment period. Selected organs were weighed.

 Results:

No mortality occurred (control, 100, 300 or 1000 mg/kg bw/day) during the observation period. The substance caused clinical signs – salivation, nuzzling up the bedding material, softer than normal stool – in male or female animals at 1000 mg/kg bw/day with short duration and degree. Therefore these were considered to be toxicologically not relevant. The body weight of the male and female animals were not adversely affected in any test item treated groups (100, 300 or 1000 mg/kg bw/day) throughout the entire observation period. The mean daily food consumption of male and female animals was comparable in male and female animals and in the test item treated animals. There were no test item related changes in the examined hematological, blood coagulation or clinical chemistry parameters in male or female animals administered with 1000, 300 or 100 mg/kg bw/day dose of the test item. Specific macroscopic alterations of the organs or tissues and changes in the investigated organ weight related to the test item were not detected. Under the conditions of the study, the test item did not cause adverse effects in male or female Hsd.Han:Wistar rats during the course of a consecutive 14 days oral administration at 100, 300 or 1000 mg/kg bw/day. At 1000 mg/kg bw/day, clinical signs (salivation, nuzzling up the bedding material, softer than normal stool), were considered to be related to test item in male or female animals. Clinical signs were with short duration and were considered to be non-toxic. There were no test item related changes at 100 or 300 mg/kg bw/day. Based on these results, a NOAEL of 1000 mg/kg bw/day was determined.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) 1272/2008 (CLP). As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008 as amended for the tenth time in Regulation(EU) No 2017/776.