Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
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 with 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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid: Clear colourless
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)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
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:
Male animals: 11-12 weeks (75-85 days) old
Female animals: 11-12 weeks (75-85 days) old
- Weight at study initiation:
Male animals: 347 – 394 g
Female animals: 186 – 223 g
- 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

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 20, 60 and 200 mg/mL. The pH of the formulations was adjusted to pH= 3-4 with 10 N NaOH. Formulations were prepared in the formulation laboratory 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurs or 14 days have elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female will be caged individually.
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 were measured on 2 occasions, during the first and last treatment weeks. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.
Concentration of the test item in the dosing formulations varied in the range of 96 and 108% of the nominal values at both analytical occasions.
Duration of treatment / exposure:
Male animals were dosed for 50 days (14 days pre-mating and 14 days mating plus an optional extended post-mating period)
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 13-18 (altogether for 50-65 days depending on day of mating)
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
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

Examinations

Parental animals: Observations and examinations:
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.
Estrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks. Animals exhibiting typical 4-5 days cycles will included in the study preferably. Vaginal smears were prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.
Vaginal smear were prepared on the day of the necropsy.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- testis weight, epididymis weight, prostate weight and seminal vesicles with coagulating glands as a whole
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded

PARAMETERS EXAMINED
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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
CLINICAL PATHOLOGY
Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry. In addition, blood samples were collected for possible determination of serum levels of thyroid hormones (T4).

HEMATOLOGY
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
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)

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

GROSS NECROPSY
- 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 / ORGAN WEIGHTS
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.
Postmortem examinations (offspring):
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.
Reproductive indices:
see table 1
Offspring viability indices:
see table 2

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, 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
Description (incidence and severity):
Functional observations did not demonstrate any test item related adverse changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were 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):
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.

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
There were no significant differences between the control and treated groups in the mean number of cycles, mean length of cycles, mean number of days in estrous or diestrus during the pre-experimental or pre-mating period. In the pre-mating period statistical significance was noted with respect to the control for the higher mean number of days in pro-estrous at 100, 300 and 1000 mg/kg bw/day. These slight differences between the control and test item administered animals was considered to be indicative of biological variation and have no toxicological significance.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 100, 300 or 1000 mg/kg bw/day. The percentage of pregnant females was statistically significantly higher than in the control group at 100, 300 and 1000 mg/kg bw/day. As a consequence, statistical significance was also noted at the higher mean fertility indices of male and female animals at 100, 300 and 1000 mg/kg bw/day with respect to their control. The gestation index was slightly lower than in the control group in female animals at 300 mg/kg bw/day as one pregnant (1/10) of this group had no pup only implantation sites. These statistical differences between the control and test item treated groups were considered to be indicative of biological variation and not related to the test item.

Details on results (P0)

The number of pregnant females and dams delivered were lower in the control group than in each test item treated groups. Duration of pregnancy, and pups stillbirths and live birth index were comparable in all groups.Statistical significance was noted with respect to the control for the higher mean number of implantations at 100 and 1000 mg/kg bw/day as well as for the higher mean number of pups at birth (total, live, viable) at 100 and 1000 mg/kg bw/day. The values of test item treated groups met well the historical control while control data were below the historical control. Therefore statistical differences were considered to have no toxicological relevance but indicative of biological variation.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of offspring with signs (cold, not suckled) at 100, 300 and 1000 mg/kg bw/day was higher than in the control group on post-natal day 0. However, these findings were not considered to be toxicologically relevant as no dose relevance was observed and the signs were transient – detected shortly after the delivery. Moreover, these observations were not associated with any influence on the development of the offspring.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
The mean number of dead offspring per litter was comparable in the control and test item treated groups on post-natal day 0 and between post-natal day 0 and 13.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean litter weight was relatively low in the control group on post-natal day 0 due to the low number of liveborns in two litters (mean pup weights were comparable). Two litters with very low pup number in control group ceased by post-natal day 4 (pups died), then the mean litter weights were similar on post-natal days 4 and 13 in all groups.
Considering the offspring’s body weight in males and females, separately, statistically significant difference with respect to the control was detected for the lower male and female pup weight at 100 and 1000 mg/kg bw/day on post-natal day 4. These differences with respect to the control in the body weight of offspring only by genders were considered to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic changes in the organs or tissues of stillborn offspring (2/2 in the control, and 1/1 at 100 mg/kg bw/day) subjected to necropsy on post-natal day 0. There were no macroscopic changes in the organs or tissues of dead pup (1/1) in the control on post-natal day 0. At 100 mg/kg bw/day, one pup (1/1) was partially cannibalized on post-natal day 7. There were no macroscopic changes in the organs or tissues of dead pup (1/1; milk in the stomach) at 300 mg/kg bw/day on post-natal day 4.
At 1000 mg/kg bw/day, there were no macroscopic findings in dead pups on post-natal day 0 (2/3) or on post-natal day 2 (1/3). Lack of the milk in the stomach was noted for one pup found dead on post-natal day 0. Hydronephrosis was observed in the left kidney of one male pup at 1000 mg/kg bw/day which was subjected to terminal necropsy on post-natal day 14. Histological examination revealed multiple cortical renal cysts as a developmental disorder without toxicological relevance.
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined
Description (incidence and severity):
There were no statistically significant differences with respect to the control in the thyroid hormone (free T4 and TSH) levels in PN13 offspring at any dose levels.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The mean number of liveborn pups (total and mean) per litter was relatively low in the control group in this study (7.0 pups/ litter). This value is below the historical control (mean: 10.9 pups/ litter, SD: 2.7, n=142). This low control value resulted in statistically significant differences in some parameters in the test item treated groups as follows: higher mean number of liveborns at 100 and 1000 mg/kg bw/day, higher mean viable pups on post-natal day 0 and 4 at 100 and 1000 mg/kg bw/day. However these values were normal and met well the historical controls.
The mean number of viable pups per litter was comparable in all groups on post-natal day 13 after litter size adjustment.
There were no significant differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices.

In male pups, statistical significances were observed at the slightly shorter mean anogenital distances (absolute and normalized) at 100, 300 and 1000 mg/kg bw/day. These alterations were in compliance with the body weight alterations (higher mean male pup weights on post-natal day 4 in the control group) and were considered to be not related to the test item. In female pups, the anogential distances were comparable in the control and test item treated groups. Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on post-natal day 13.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: TABULAR SUMMARY

 

VALUES PER GROUP

OBSERVATIONS

Control

 

 

 

 

100

mg/kg

bw/day

300

mg/kg

bw/day

1000

mg/kg

bw/day

 

 

Pairs started (n)

12

12

12

12

Estrous cycle - mean length (days)*

4.3

4.2

4.2

4.3

- frequency of irregular cycled**

6/12

2/12

4/12

2/12

Females showing evidence of copulation (n)

12

12

12

12

Females achieving pregnancy (n)

3

11

10

10

Conceiving days 0 - 5 (n)

3

11

10

9

Conceiving days 6 - 7 (n)

0

0

0

1

Pregnancy < 21 days (n)

2

5

4

6

Pregnancy = 22 days (n)

5

6

4

3

Pregnancy > 23 days (n)

1

0

1

1

Dams with live young born (n)

3

11

9

10

Dams with live young at day 4 pp (n)

5

11

9

10

Implants dam (mean; n)

7.9

12.5

10.6

12.4

Live pups/dam at birth (mean; n)

7.0

11.2

9.7

11.5

Live pups/dam at day 4 (mean; n)

6.5

11.2

9.6

11.2

Sex ratio (m/f) at birth (mean; n/n)

3.3/ 3.8

5.7/5.5

4.8/4.9

5.6/5.9

Sex ratio (m/f) at day 4 (mean; n/n)

3.3/3.3

5.7/5.5

4.7/4.9

5.4/5.8

Litter weight at birth (mean; g)

49.8

66.1

59.7

69.0

Litter weight at day 4 (mean; g)

113.7

108.9

100.1

113.6

Pup weight at birth (mean; g)

6.2

6.0

6.1

6.1

Pup weight at day 4 (mean; g)

11.0

10.1

10.4

10.3

Pup weight at day 4 (mean; g; m f)

11.3/10.5

10.0/9.7

10.8/10.2

10.4/9.9

Normalized anogenital distance at day 4 mean; mm; m/f)

3.0/1.7

2.8/1.7

2.8/1.7

2.9/1.7

Pup weight at day 13 (mean; g)

29.5

29.1

29.6

29.7

Number of nipples - male pups at day 13 (mean; n)

0

0

0

0

ABNORMAL PUPS

Dams with 0

0

0

0

0

Dams with 1

0

0

0

0

Dams with > 2

0

0

0

0

LOSS OF OFFSPRING

Pre-natal/ post-implantation (implantations minus live births)

Females with 0

3

5

5

5

Females with 1

3

2

1

3

Females with 2

2

2

2

0

Females with > 3

0

2

1

2

Post-natal (live births minus alive at post-natal day 13)

Females with 0

5

10

5

5

Females with 1

2

0

1

1

Females with 2

1

1

0

1

Females with > 3

0

0

0

0

Remarks:      

* = estrous cycle examined during the pre-mating (treatment) period

**= frequency of irregular cycle = number of animals with irregular cycle/number of animals examined

n = number of dams or pups

m = male

f = female

g =gram

 

Applicant's summary and conclusion

Conclusions:
Based on the results the NOAEL for systemic toxicity of male/female rats was 1000 mg/kg bw/day, the NOAEL for reproductive performance of male/female rats was 1000 mg/kg bw/day and the NOAEL for F1 Offspring was 1000 mg/kg bw/day.
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according OECD TG 422 to obtain initial information on the toxic potential of the test item and its possible effects on male and female reproductive performance as well as development of the F1 offspring from conception to day 13 post-partum. The test item was repeatedly administered to parental animals at doses of 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day.

Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) 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.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. Test item concentrations in the dosing formulations varied in the acceptable range between 96 % and 108 % of the nominal values confirming the proper dosing.

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). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.

The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

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 andin 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; haemorrhage, 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.

Results:

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 behaviour 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).

A test item influence on the estrous cycle was not found at any dose level (100, 300 and 1000 mg/kg bw/day).

There were no test item related differences between the control and test item treated groups in the reproductive performance of male and female animals.

There were no toxicologically relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (100, 300 and 1000 mg/kg bw/day).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 coagulatinggland) 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 offspring’s development was not adversely influenced at any dose level.

Under the conditions of the study, 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.

The test item did not adversely influence the reproductive performance or fertility (gonad function, mating behavior, conception, parturition) in parental male and female rats at the doses of 100, 300 and 1000 mg/kg bw/day.

The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams.

Based on the results the NOAEL for systemic toxicity of male/female rats was 1000 mg/kg bw/day, the NOAEL for reproductive performance of male/female rats was 1000 mg/kg bw/day and the NOAEL for F1 Offspring was 1000 mg/kg bw/day.