5',5'''-[ethylenebis(p-phenyleneazo)]bis[1',2'-dihydro-6'-hydroxy-4'-methyl-2'-oxo-1,3'-bipyridinium] dilactate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 29th to December 07th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 29 July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Basic Yellow 094

IUPAC Name:

Basic Yellow 094

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest.
- Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study. Only healthy animals were used for the study. Healthy status was certified by the breeder.
- Females: nulliparous and non-pregnant.
- Age at study initiation: 90 – 92 days.
- Weight at study initiation: males 345 – 433 g ; females 198 – 242 g. The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: before mating: 2 animals of the same sex/cage; during the mating 1 male and 1 female / cage. Pregnant females were individually house, while males after mating were housed 2 animals / cage. Type III polypropylene/polycarbonate (22 × 32 × 19 cm).
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice, ad libitum. Food was changed at weekly intervals.
- Water: tap water, ad libitum. Fresh drinking water was given daily.
- Acclimation period: 27 days.

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod: artificial light, from 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

FORMULATION
Formulations were prepared beforehand not longer than four days and stored at room temperature until use.

TREATMENT
- Volume: a constant treatment volume of 5 ml/kg body weight was administered in all groups.
- Administration: the individual volume of the treatment was based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on Day 0.

DOSES CHOICE
The dose levels were chosen on the basis of the results of a preliminary dose range finding study.

Details on mating procedure:

Mating begun 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage. Females remained with the same male until copulation occurred or 14 days have elapsed.
For three female animals their male pair was replaced by a proven male within the same group and after the 14 days mating period elapsed to ensure the appropriate number of pregnants.
Vaginal smears were examined for the presence of vaginal plug or sperm.
Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy). Sperm positive females were caged individually.
Mating pairs were clearly identified in the raw data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations was performed twice during the study. Five aliquots of each formulation and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis.
Concentration of the test item in the dosing formulations varied between the range of 100 % and 106 % in comparison to the nominal values.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 98 % at ca. 1 mg/ml and 98 % at ca. 200 mg/ml).
Test item proved to be stable in distilled water at the intended concentrations at room temperature for four days.

Duration of treatment / exposure:

42 days (males)
42-65 days (females, depending on the efficiency of mating)

Frequency of treatment:

7 days/week

Details on study schedule:

Dosing of both sexes begun after 27 days acclimatization – including 13 days pre-treatment estrous cycle examination – and was continued up to and including the day before the necropsy.
The mating phase started after 14-days treatment (pre-mating) period.
Male animals were dosed for 42 days (14 days pre-mating and 1-16 days mating plus 12-27 days of post-mating period; until the necessary number of pregnant female animals was evident); then they were sacrificed.
Females were dosed for 14 days pre-mating, through 1-16 days mating period and throughout pregnancy and at least up to and including day 12 post-partum or the day before sacrifice.
The day of birth (viz. when parturition is complete) was defined as day 0 post-partum.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

83 mg/kg bw/day (actual dose received)

Remarks:

as active ingredient, corresponding to 100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

as active ingredient, corresponding to 300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

834 mg/kg bw/day (actual dose received)

Remarks:

as active ingredient, corresponding to 1000 mg/kg bw/day

No. of animals per sex per dose:

12 animals/sex in the control and dose groups

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

MORTALITY
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS
General clinical observations were made on parental animals once a day, after the administration at approximately the same time.
Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy.
Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
Observations were performed 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 was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

NEUROBEHAVIOURAL EXAMINATION
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behavior of animals were tested. A modified Irwin test was performed.

BODY WEIGHT
The body weight of all parental animals was determined with an accuracy of 1 g. Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy. 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 weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not valuated statistically.
Additionally, the body weight of female animals was determined daily because of body weight loss and for ensuring a propped dosing. These values were recorded bot not reported.
Body weight was measured on day of necropsy for female animals subjected to organ weighing (selected for further examinations).
Body weight data were reported individually for adult animals. Individual body weight change was calculated.

FOOD CONSUMPTION
The food consumption was determined weekly by reweighing the given and non-consumed diet with an accuracy of 1 g during the treatment period except mating phase (pre-mating days 7, 13, and post-mating days 20, 27, 34 and 41 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 12 or 13 for female animals).

CLINICAL CHEMISTRY
Clinical pathology examinations 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 CP® anesthesia.
Parameters: Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Urea concentration (UREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Sodium concentration, Potassium concentration, Albumin concentration (ALB), Total Protein concentration (TPROT).

HAEMATOLOGY
Blood samples were collected 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 CP® anesthesia.
Parameters: White Blood Cell (leukocyte) count (WBC), Red Blood Cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (relative volume of erythrocytes, HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) Hemoglobin (MCH), Mean Corpuscular (erythrocyte) Hemoglobin Concentration (MCHC), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count, Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT).

THYROID HORMONES
Examinations 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 CP® anesthesia.
Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH).
Blood samples were collected from all dams on post-partal/post-natal day 13 and from all parent male animals at termination on Day 42.
Parameters were measured: Thyroxine – free Tetra-iodothyronine (FT4), Thyroid-stimulating hormone (TSH).

EXAMINATION OF PLACENTAL SIGN
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If the test was negative on day 13, the examination was repeated on day 14 of gestation.

OBSERVATION OF DELIVERY PROCESS
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible from gestational day 21 onwards. All observations and any evidence of abnormal deliveries were considered. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their new-born or not. The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
On post-partum day 4, the size of each litter was adjusted to four pups per sex per litter, if it was feasible. Extra pups were eliminated by a random selection. Partial adjustment of litter size was performed if the number of male and female pups did not allow having four of each sex per litter (for example, five males and three females).

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization.
Vaginal smears were also 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 mating. Vaginal smears were also prepared on the day of the necropsy. Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.

Litter observations:

OBSERVATION POST-DELIVERY
Each litter was examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition is complete, day 0), and on days 4 and 13 post-partum with an accuracy of 0.1 g. Any abnormal behavior of the offspring was recorded.
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups).
All litters were checked and recorded daily for the number of viable and dead pups. Dead pups found were subjected to necropsy by macroscopic examination. All observed abnormalities were recorded.

BODY WEIGHT
Individual body weight of pups was also determined with an accuracy of 0.01 g on postnatal day 4 and the litter weight was calculated for evaluation on postnatal day 4.

ANOGENITAL DETERMINATION, NIPPLES OBSERVATIONS
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight. The number of nipples/areolae in male pups was counted on postnatal day 13.

THYROID HORMONES
Examinations 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 CP® anesthesia.
Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH).
Blood samples were collected from 2-7 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter) and from 1-7 pups per litter on post-partal/post-natal day 13.
Parameters were measured: Thyroxine – free Tetra-iodothyronine (FT4), Thyroid-stimulating hormone (TSH).

Postmortem examinations (parental animals):

SACRIFICE
All animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP®. All animals were subjected to gross necropsy as follows: parental male animals after the post-mating period on Day 42; dams on post-partum day 13 or shortly thereafter (between Days 51 and 65); 2 dams at 834 mg/kg bw/day were euthanized on lactation day 6 as no living pups remained; non-pregnant and not mated females on Days 42 or 51.

GROSS PATHOLOGY
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
The ovaries, uterus with cervix and oviduct, vagina, testes, epididymides (total and cauda), prostate and seminal vesicles with coagulating glands, adrenal glands and pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides) for five male and five female animals randomly selected from each group: adrenal glands, aorta, bone with bone marrow and joint (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (lachrymal gland with Harderian glands), gonads (testes with epididymides, ovaries, uterus with fallopian tube and vagina), 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), mammary gland, 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 and urinary bladder.

ORGAN WEIGHT
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
The thyroid weight will be determined – if relevant – after fixation.
Paired organs were weighed together.

HISTOPATHOLOGY
Detailed histological examination was performed on the ovaries, uterus with cervix and oviduct, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Histological examination of these organs was performed in non-pregnant and not mated female animals and their mating partner in the low and mid dose groups (1/12 male at 83 mg/kg bw/day; 3/12 male and 2/12 female at 250 mg/kg bw/day).
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (834 mg/kg bw/day) groups.
In addition, liver, spleen, kidneys, uterus with cervix and oviduct, vagina, skin and stomach were processed histologically in animals showing macroscopic findings in these organs at the necropsy: skin 2/12 male at 83 mg/kg bw/day; kidneys 1/12 female at 83 mg/kg bw/day; 1/12 male and 1/12 female at 250 mg/kg bw/day; liver 2/12 female at 250 mg/kg bw/day; spleen 3/12 female at 83 mg/kg bw/day, 1/12 male and 2/12 female at 250 mg/kg bw/day; stomach 1/12 male at 250 mg/kg bw/day; uterus 1/12 female at 250 mg/kg bw/day; sexual organs of one male and one female animal at 250 mg/kg bw/day were processed due to technical mistake.
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Postmortem examinations (offspring):

SACRIFICE
All animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP®. All animals were subjected to gross necropsy, offsprings on postnatal day 13 or shortly thereafter.

GROSS PATHOLOGY
Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.

Statistics:

The statistical evaluation of appropriate data was 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) is 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.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Reproductive indices:

- Copulatory index: measure of animals’ ability to mate.
- Fertility index: measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
- Gestation index: measure of pregnancy that provides at least one live pup.

Offspring viability indices:

- Post-implantation mortality (intrauterine mortality).
- Post-natal mortality.
- Survival Index.
- Sex ratio.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of systemic toxicity related to the test item were detected in some animals at 250 mg/kg bw/day (piloerection, decreased activity and hunched back in single male) and at 834 mg/kg bw/day (piloerection, decreased activity in two male animals and in one dam, noisy breathing or diarrhea activity in one male and in one dam) dose levels at the daily and weekly clinical observations.
The stool was soft in each male and female animal at 83 mg/kg bw/day from Day 5 and paler than normal and soft in each male and female animal at 250 and 834 mg/kg bw/day during the entire observation period (from Day 1 up to and including last day of the treatment).
The color of stools was indicative of presence of the test item or its metabolite(s) in the gastro-intestinal tract.
The control male animals exhibited normal behavior and physical condition with no abnormalities at the daily or at the detailed weekly clinical observations.
At 83 mg/kg bw/day, soft stool was observed in the bedding material for each male animal from Day 5 up to the last treatment day (Day 41). Alopecia was detected in two male animals at 83 mg/kg bw/day (2/12) on the forelimbs, scrotum and thigh (1/12) and between the ears and under the right ear (1/12) daily from Day 13 to Day 41 and at the weekly clinical observations.
At 250 mg/kg bw/day, decreased activity, piloerection and hunched back were observed in one male animal (1/12) daily from Day 23/27 to Day 33 or 41 and weekly at the detailed observations. Paler than normal and soft stool was seen in the bedding material of male animals (12/12) from Day 1 to termination of the treatment.
Paler than normal and soft stool (12/12) was observed in male animals at 834 mg/kg bw/day during the entire treatment period (from Day 1 to Day 41). Piloerection (2/12), decreased activity (2/12), noisy breathing (1/12) and diarrhea (1/12) were observed in two male animals at 834 mg/kg bw/day at the daily and at the weekly clinical observations.
There were no clinical signs in female control animals during the entire observation period (pre-mating, post-mating, gestation and lactation periods).
In the female animals, soft stool (12/12 at 83 mg/kg bw/day), paler than normal and soft stool (12/12 at 250 and 834 mg/kg bw/day) were observed from Day 1 and Day 5 respectively, up to the termination of the study (pre-mating, post-mating, gestation or lactation periods).
One non-pregnant female at 250 mg/kg bw/day showed piloerection and decreased activity daily between Days 27/28 and 37, and at the weekly clinical observations on Days 28 and 35.
One dam at 834 mg/kg bw/day showed piloerection and decreased activity daily during lactation days 2-5 and at the detailed weekly clinical observation on lactation day 4.
The color of stools was indicative of presence of the test item or its metabolite(s) in the gastro-intestinal tract.

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality at 83, 250 or 834 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was depressed in male animals at 83, 250 and 834 mg/kg bw/day during the entire treatment period and in female animals at 83, 250 and 834 mg/kg bw/day during the first week of the treatment and during the gestation period. However, at 83 mg/kg bw/day the difference with respect to the control in the mean body weight was low (< 10 %) in male and female animals, thus, the toxicological relevance of the finding at this dose level is questionable.
Statistical significance with respect to the control was detected at the slightly lower mean body weight of male animals at 83 mg/kg bw/day on Days 20 and 41, at 250 and 834 mg/kg bw/day from Day 7 up to the termination the study (on Days 7, 13, 20, 27, 34 and 41).
The mean body weight gain was below the control value in male animals at 83, 250 and 834 mg/kg bw/day in the most cases reaching statistical significances and in several case during the treatment period: between Day 0-7, 7-13, 13-20 and between Days 0 and 41 at 83 mg/kg bw/day; between Days 0-7, 13-20, 20-27, 27-34 and between Days 0 and 41 at 250 mg/kg bw/day; between Days 0-7, 27-34 and between Day 0 and 41 at 834 mg/kg bw/day.
The mean body weight was comparable in the control and at 83 and 250 mg/kg bw/day groups in female animals during the pre-mating period; slightly lower mean body weight was observed in female animals at 834 mg/kg bw/day compared to the control on Day 7 during the pre-mating period when compared to the control.
During the gestation period, the mean body weight of dams was lower than in the control group reaching statistical significances at 83 mg/kg bw/day on gestation day 21, at 250 and 834 mg/kg bw/day on gestation days 7, 14 and 21.
The mean body weight of dams was also lower than in the control group at 83 mg/kg bw/day on lactation day 0 and at 250 and 834 mg/kg bw/day on lactation days 0, 4 and 13.
There were no statistical differences in the mean body weight gain between the control and 83 or 250 mg/kg bw/day groups of female animals during the pre-mating period. Statistical significance was observed at the slightly lower mean body weight gain (between Days 0 and 7) and at the higher mean body weight gain (between Days 7 and 13) of female animals administered with 834 mg/kg bw/day comparing to the control.
The mean body weight gain was lower than in the control group during the gestation period (between gestation day 0 ad 21) at 83 mg/kg bw/day, at 250 mg/kg bw/day (between gestation days 0-7, 7-14, 14-21, 0-21) and at 834 mg/kg bw/day (between gestation days 7-14, 14-21 and for the study overall). During the lactation period, the mean body weight gain was similar in the control and test item administered animals at 83, 250 and 834 mg/kg bw/day groups. Although, the body weight of one dam significantly decreased after delivery at 834 mg/kg bw/day (between lactation days 0 and 4).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption was significantly reduced in male and female animals at 250 and 834 mg/kg bw/day.
The mean daily food consumption was comparable in the control and at 83 mg/kg bw/day in male animals during the entire treatment period.
Statistically significant difference with respect to the control was detected at the lower mean food consumption in male animals at 250 mg/kg bw/day between Days 0 and 7 and at 834 mg/kg bw/day between Days 0 and 7 and between Days 27 and 34.
The mean daily food consumption of female animals was lower than in the control group in the most cases during the pre-mating and gestation period at 83, 250 and 834 mg/kg bw/day reaching statistical significances in several cases: at 83 mg/kg bw/day during the entire pre-mating period and between gestation days 7 -14, 14-21; at 250 mg/kg bw/day between pre-mating days 0-7, between gestation days 7-14, 14-21 and at 834 mg/kg bw/day between pre-mating days 0-7, between gestation days 14-21.
During the lactation period, the mean daily food consumption was similar to the control at 83 mg/kg bw/day and statistical significance was detected at the lower mean food consumption at 250 and 834 mg/kg bw/day between lactation days 4 and 13.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological evaluation revealed changes in percentage of reticulocytes in female animals at 83, 250 and 834 mg/kg bw/day along with changes in red blood cell parameters (red blood cell count, hemoglobin concentration, mean corpuscular volume and mean corpuscular hemoglobin content) at 834 mg/kg bw/day.
Slight elevation of platelet count in male animals at 834 mg/kg bw/day might be related to the test item effect. However, there were no accompanying changes in the investigated parameters (PT, APTT), therefore toxicological importance of this findings is questionable.
All examined hematological and blood coagulation parameters were comparable with the control in male animals at 83 and 250 mg/kg bw/day.
At 834 mg/kg bw/day, the mean platelet count (PLT) was slightly but statistically significantly higher than in the control group in male animals.
In the female animals at 83 mg/kg bw/day, statistically significances were observed at the slightly higher mean corpuscular volume (MCV), at the higher mean percentage of reticulocytes (RET) and at the slightly shorter mean prothrombin time (PT) when compared to the control.
At 250 mg/kg bw/day, higher mean percentage of lymphocytes (LYM), lower mean percentage of monocytes (MONO), lower mean corpuscular hemoglobin concentration (MCHC), shorter mean prothrombin time and significantly higher mean percentage of reticulocytes were detected with respect to their control in female animals.
In female animals at 834 mg/kg bw/day, with respect to their control, lower mean percentage of monocytes, mean red blood cell count (RBC), hemoglobin concentration (HGB) and mean corpuscular hemoglobin concentration as well as higher mean corpuscular volume, higher mean corpuscular hemoglobin content (MCH) and significantly elevated mean percentage of reticulocytes were observed.
Slight elevation of platelet count in male animals at 834 mg/kg bw/day was mainly due to the significantly higher number of platelet count in one animal (no. 411) along with slightly elevated white blood cell count (WBC) and percentage of neutrophil granulocytes and reduced percentage of lymphocytes. A test item influence cannot be excluded entirely, however, there were no accompanying changes in the investigated parameters (PT, APTT) as platelet maintain the hemostasis. Therefore, toxicological importance of this findings is questionable.
The elevated mean percentage of reticulocytes in female animals at 83, 250 and 834 mg/kg bw/day was in connection with the test item effect along with changes of some red blood cell parameters mainly at 834 mg/kg bw/day (MCV, RBC, HGB, MCH and MCHC).
Other changes in female animals at 83 and 250 mg/kg bw/day – with respect to their control – were with minor degree or not related to doses (LYM, MCV, MCHC, PT). Therefore, these findings in female animals were considered to be toxicologically not relevant.
Monocytopenia – detected at 250 and 834 mg/kg bw/day – usually has no diagnostic significance.

SERUM THYROID HORMONE LEVELS
Statistical significances with respect to the control was detected at the lower mean FT4 level in the parental male animals at 83, 250 and 834 mg/kg bw/day independently from doses. There were no changes in the TSH levels of these animals.
In the female animals, statistical significances were noted for the lower mean FT4 levels at 83.4 and 834 mg/kg bw/day when compared to the control. There were no changes in the TSH levels of these animals

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 83, 250 or 834 mg/kg bw/day (male or female).
In male animals, statistical significances with respect to the control were observed at the slightly lower mean sodium (Na+) concentration at 83 mg/kg bw/day and at the slightly lower mean concentration of glucose (GLUC) at 250 g/kg bw/day.
Slight changes in total bilirubin concentration in female animals at 834 mg/kg bw/day might be related to changes in red blood cell parameters. However, values were well within the historical control range.
There were no statistically significant differences between the control and 834 mg/kg bw/day treated male animals at the investigated clinical chemistry parameters. However, the total bilirubin (TBIL) level was markedly elevated in male animal showing changes in PLT, WBC, LYM and NEU at 834 mg/kg bw/day.
In the female animals at 83 mg/kg bw/day, the mean concentration of sodium was lower than in the control group.
In the female animals administered with 250 mg/kg bw/day, statistical significance with respect to the control was observed at the higher mean concentration of cholesterol (CHOL) the lower mean creatinine concentration (CREA).
The mean concentration of total bilirubin and cholesterol was above the control value in female animals at 834 mg/kg bw/day.
Changes in concentration of total bilirubin were related to doses in female animals at 83, 250 and 834 mg/kg bw/day, reaching statistical significance at the high dose only. However, the individual values remained well within the historical control ranges.
The statistically significant differences in Na+, GLUC, CREA, CHOL with respect to their controls were considered to have little or no toxicological importance because values – mean and individual – corresponded to the historical control values, there was no dose relevance or related histopathological alterations.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 83, 250 or 834 mg/kg bw/day groups at the end of the treatment period (on Day 42 for male animals and on Day 48 for female animals).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Alopecia, was noted for one male animal (1/5 at 83 mg/kg bw/day) between ears and under right ear – approximately 2 cm in width – at the functional observations.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test item related lesions detectable by histological examination of the investigated reproductive organs of male animals at 83, 250 or 834 mg/kg bw/day.
Histopathological examinations revealed test item related chronic progressive nephropathy (CPN) and hyperplasia in the spleen (hyperplasia of hematopoietic erythroid, myeloid and lymphoid cells) in female animals at 83, 250 or 834 mg/kg bw/day.

In the male animals belonging to the control and treated groups (12/12 control; 1/1 at 83 mg/kg bw/day, 4/4 at 250 mg/kg bw/day, 12/12 at 834 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases. 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 prostate, epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (12/12 control; 3/3 at 250 mg/kg bw/day, 12/12 at 834 mg/kg bw/day, including non-pregnant or not mated females), the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Dilatation of uterine horns was observed in one dam at 250 mg/kg bw/day (1/4). 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.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
Histological examination revealed chronic progressive nephropathy (CPN) in several animals – 1/5 control female; 1/1 male at 250 mg/kg bw/day; 1/5 male and 6/8 female at 834 mg/kg bw/day – in different severity. CPN is an important spontaneous renal disease of the commonly used strains of laboratory rat because it is a serious confounding factor in experimental pathology and toxicology studies. In this study – regarding the higher incidence of CPN in the animals belonging to the 834 mg/kg bw/day treated female group – it cannot be excluded that the high dose of test item was a predisposing factor in the pathogenesis of this renal lesion. There are no details for the explanation of higher incidence of CPN in the female animals as compared to the males.
Hyperplasia – associated with enlargement – of spleen was detected in test item treated groups, mainly in female animals: 1/1 male at 834 mg/kg bw/day; 3/8 female at 83 mg/kg bw/day, 2/7 female at 250 mg/kg bw/day and 11/11 female 834 mg/kg.
The hyperplasia affected all the hematopoietic cells (erythroid, myeloid and lymphoid) in the spleen. In this study, hyperplasia affected only treated animals and was considered to be treatment related lesion. Histological results gave no explanation on the higher incidence in the female animals as compared with the male groups.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 male and 1/5 female in the control, 1/5 female at 834 mg/kg bw/day), were detected sporadically. The pulmonary emphysema is considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (3/5 control male; 1/5 female at 834 mg/kg bw/day) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Erosion in the stomach (1/1 male at 250 mg/kg bw/day) was considered an individual disease.
Focal fibrosis in the Glisson’s capsule of the liver (2/2 female at 250 mg/kg bw/day) may be the consequence of the mechanical irritation due to the diaphragmatic hernia.
Pyelectasia (one or both sides) was observed in several animal: 1/5 male and 2/5 female control; 1/1 female at 83 mg/kg bw/day; 1/1 male and 1/1 female at 250 mg/kg bw/day and 2/5 male and 2/8 female at 834 mg/kg bw/day. This finding without other pathological lesion (degeneration, inflammation, fibrosis) is a common finding in Wistar rats without toxicological significance.
Focal atrophy of hair follicles (reduced in size) in two male animals at 83 mg/kg bw/day (2/2) without inflammatory was in connection with the focal alopecia observed at the necropsy. Abnormal hair follicle atrophy might be due to inflammatory lesion, dermal ischemia or autoimmune conditions, as well.
No morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the cardiovascular system, the respiratory system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Other effects:

no effects observed

Description (incidence and severity):

DELIVERY DATA OF DAMS
There were no toxicologically relevant differences in the evaluated parameters of delivery between the control and test item treated groups (83, 250 or 834 mg/kg bw/day).
The mean number of implantation sites per dams and the mean of post-implantation loss were comparable in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the mean duration of pregnancy, in the mean number of total births, liveborns, stillborns or viable pups on post-partal day 0, in the percentage of dams with viable offspring on post-partal day 0 or in the live birth index.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A clear evidence of the test item influence on the estrous cycle was not detected at any dose level (83, 250 or 834 mg/kg bw/day).
There were no statistically or biologically significant differences between the control and 83 mg/kg bw/day treated group in the examined parameters of estrous cycle.
At 250 mg/kg bw/day, statistical significance was noted for the slightly lower mean number of days in pro-estrous and higher mean number of days in diestrous when compared to the control.
At 834 mg/kg bw/day, lower mean number of cycles, days in pro-estrous and estrous, higher mean number of days in diestrous were detected when compared to the control.
The mentioned differences with respect to their controls in the investigated parameters of estrous cycle during the premating period were similar to those during the pre-experimental period therefore these slight differences were considered to be of slight or no toxicological significance.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not affected by the test item at 83, 250 or 834 mg/kg bw/day in male or female animals.
There were statistical differences between the control and the 83 mg/kg bw/day groups in male animals at the lower copulatory index (lower percentage of mated males) and at the higher percentage of fertility index (percentage of fertile male animals).
The copulatory index was slightly lower in male animals at 250 mg/kg bw/day comparing to the control.
The examined parameters of reproductive performance were similar in male animals the control and 834 mg/kg bw/day groups.
In the female animals, statistical significances were noted for the lower percentage of non-pregnant females, higher percentage of pregnant females and higher fertility index at 83 mg/kg bw/day and at the lower copulatory index at 250 mg/kg bw/day.
There was no difference between the control and 834 mg/kg bw/day treated groups in the number of pregnant females and dams delivered, in the fertility indices, gestation index, in the pre-coital interval and conceiving days.

Details on results (P0)

Two dams at 834 mg/kg bw/day (no. 426 and 422), were subjected early necropsy on lactation day 6. Therefore, procedures and observations of in-life period for these dams were terminated on lactation day 5.
Two dams at 250 mg/kg bw/day (no. 329 and 331) had to be subjected to necropsy on lactation day 13, therefore, procedures and observations of in-life period for these dams were terminated on lactation day 12.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

834 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

LOAEL

Effect level:

83 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

haematology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

83 mg/kg bw/day (actual dose received)

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs were detected in a higher percentage with respect to the control in the offspring at 250 and 834 mg/kg bw/day between postnatal days 0 and 13.
At 83 mg/kg bw/day, the percentage of not suckled pups (no milk in the stomach) was lower and percentage of cold and dead pups were similar to those in the control group.
At 250 mg/kg bw/day, the percentage of not suckled pups was slightly higher than in the control group. Decreased activity and missing of all pups were detected in the litter of one dam.
At 834 mg/kg bw/day, cold body temperature, no milk in the stomach, cachexia, death and missing pups were observed with higher percentage when compared to the control groups.

The above-mentioned clinical signs of offspring were probably mainly due to the lack of nursing or bad conditions of some dams at 250 and 834 mg/kg bw/day.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The extrauterine mortality of offspring was higher than in the control group at 834 mg/kg bw/day presumably due to the inadequate nursing by mothers.
Statistical significance was detected at the higher mean number of dead (including missing) pups at 834 mg/kg bw/day with respect to the control between post-natal days 0 and 13.
The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were no significant differences between the control and test item treated (83, 250 or 834 mg/kg bw/day) groups in the survival indices.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development of the offspring was depressed at 83, 250 and 834 mg/kg bw/day.

The mean litter weight was comparable to the control group in offspring at 83 mg/kg bw/day, although the mean litter weight gain was statistically significantly lower than in the control group between post-natal days 0 and 4 and between post-natal days 0 and 13.
The mean litter weights were significantly lower than in the control at 250 and 834 mg/kg bw/day on postnatal day 13 and at 834 mg/kg bw/day on post-natal day 4 and due to the significantly lower mean litter weight gain between post-natal days 0-4 and 4-13. The summarized litter weight gain remained also below the control at 250 and 834 mg/kg bw/day,
The mean pup weights as well as the mean pup weight gains were lower than in the control in all test item treated groups (83, 250 and 834 mg/kg bw/day) during the entire observation period reaching statistical significances in the most cases. The difference with respect to the control was with minor degree (<10 %) at 83 mg/kg bw/day, therefore this finding was judged to be toxicologically not relevant.
Considering the offspring’s body weight in males and females separately, statistically significant difference with respect to the control was detected at the lower mean weight of male and female pups at 83, 250 and 834 mg/kg bw/day on postnatal day 4.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

SERUM THYROID HORMONE LEVELS
In the offspring sampled on post-natal day 4, the FT4 and TSH concentrations were comparable in the control and test item treated groups (83, 250 and 834 mg/kg bw/day).
Sampled on postnatal day 13, the mean FT4 concentrations were below the control value at 250 and 834 mg/kg bw/day and the mean TSH concentrations exceeded the control value at 83, 250 and 834 mg/kg bw/day reaching statistical significance at 834 mg/kg bw/day.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Slight differences with respect to the control were detected at the absolute anogenital distances at 83 mg/kg bw/day (male pups) and at 250 and 834 mg/kg bw/day (male and female pups) as well as at the normalized anogenital distances at 250 and 834 mg/kg bw/day (female pups).
In the male pups, the mean absolute anogenital distance was shorter than in the control group at 83, 250 and 834 mg/kg bw/day and the normalized anogenital distances were identical in the control and treated groups therefore have no toxicological relevance.
In the female pups, statistical significance was observed at the shorter mean absolute and normalized anogenital distances with respect to the control group at 250 and 834 mg/kg bw/day independently from doses. The differences with respect to the control were in good correlation with the body weight of the pups and were with minor degree thus these findings were considered to have little or no toxicological importance.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipples/areoles were not visible in any of the examined male offspring in the control, 83, 250 or 834 mg/kg bw/day groups on postnatal day 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cachexia and empty stomach were noted for most of dead pups subjected to necropsy: 1/1 at 83 mg/kg bw/day on post-natal day 8, 1/1 at 250 mg/kg bw/day on post-natal day 4; 8/9 at 834 mg/kg bw/day on post-natal day 12 or 13.
There were no macroscopic changes in the organs or tissues of stillborn offspring (1/1 at 83 mg/kg bw/day and 1/1 at 834 mg/kg bw/day) subjected to necropsy on postnatal day 0 and in one of the dead pups at 834 mg/kg bw/day (1/9) subjected to necropsy on postnatal day 11.
Regarding the late mortality of pups (postnatal day, 4, 8, 12, 13) and empty stomach of pups, the cause of death was probably the inadequate nursing by mothers.

Other effects:

no effects observed

Description (incidence and severity):

There were no test item related differences between the control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

83 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: in presence of maternal toxicity

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL (reproductive performance) = 834 mg/kg bw/day
NOAEL (offspring) = 83 mg/kf bw/day, in presence of maternal toxicity

Executive summary:

The objective of the study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 83, 250 and 834 mg/kg bw/day by active substance content (corresponding to uncorrected doses of ca. 100, 300 and 1000 mg/kg bw/day).

The substance was administered to four groups of Han:WIST rats consisting of 12 animals per sex per group. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study; the test item concentrations in the dosing formulations varied within the range of 100 % and 106 % in comparison to the nominal values and confirming the proper preparation of the dosing formulations.

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 42 days). Dams were additionally exposed through the gestation period and up to and including lactation days 5 (no living pups remained) or 12-15, i.e. up to the day before necropsy (females were administered altogether for 42-65 days).

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for 13 days and from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.

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. Two dams were euthanized and subjected to early necropsy because no living pups remained.

Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH) from 2-7 pups per litter (where it was feasible) on post-natal day 4, from all dams and from 1-7 pups per litter (where it was feasible) at termination on post partum/post-natal day 13 and from all parent male animals at termination (day 42).

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes and epididymides and seminal vesicles with coagulating glands and prostate of all adult male animals were determined. Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus with cervix and oviduct, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female). In addition, these organs were processed histologically in not mated and non-pregnant females and their mating partner and in not mated male animals in the low or mid dose group.

Five dams and their 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 examination.

Organs showing macroscopic findings at the necropsy were also processed and examined histologically in animals of the low and mid dose groups.

There was no test item related mortality at any dose level.

A test item influence on the estrous cycle was not found at any dose level. There were no toxicologically significant differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams.

There were no test item related changes in the serum thyroid hormone levels (TSH and FT4) at any dose level in parental male animals.

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level. No toxic or other test item related lesions detectable by histological examination was found in the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) of male or female animals administered with 834 mg/kg bw/day.

As for the offspring, higher extrauterine mortality (834 mg/kg bw/day), clinical signs with higher percentage (250 and 834 mg/kg bw/day), shorter anogenital distances (250 or 834 mg/kg bw/day) and reduced body weight development (250 or 834 mg/kg bw/day), elevated TSH level along with slightly lower – not dose related – concentration of FT4 at 250 and 834 mg/kg bw/day on postnatal day 13 were seen when compared to their control.

Overall, based on the conditions of the present study, test item did not influence the reproductive performance or the delivery parameters. The F1 offspring development was influenced by test item after repeated oral administration of dams from birth to post-natal day 13. However, although several parameters of offspring were altered, these changes might be – at least partially - the consequence of the maternal toxicity, i.e. mainly due to lack or inadequate nursing by mothers or bad conditions of some dams at 250 and 834 mg/kg bw/day.

Conclusion

NOAEL for reproductive performance of male/ female rats: 834 mg/kg bw/day

NOAEL for F1 offspring: 83 mg/kg bw/day, in presence of maternal toxicity