Registration Dossier

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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 30 to August 12, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Remarks:
validated analytical method

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2019

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Fluorescent Brightener 363
IUPAC Name:
Fluorescent Brightener 363

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, Cserkesz u. 90. Hungary.
- Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
- Females: nulliparous and non-pregnant.
- Age at study initiation: 92 – 97 days parent males and females.
- Weight at study initiation: 365 – 418 g male animals and 208 – 246 g female animals. The weight variation did not exceed ± 20 per cent of the mean weight
- Animal health: only healthy animals were used for the study. Healthy status was certified by the breeder.
- Housing: 2 animals of the same sex/cage before mating; 1 male and 1 female / cage during the mating; individually pregnant females: individually; 2 animals / cage males after mating. Cage type III polypropylene/polycarbonate (22 x 32 x 19 cm).
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany -, ad libitum. Food was changed at weekly intervals. 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.
- Water: animals received tap water from watering bottles, as for human consumption, ad libitum. Food was changed at weekly intervals. Fresh drinking water was given daily. 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. The quality control results are available at testing facility's archives.
- Acclimation period: 27 days

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:
water
Details on exposure:
TREATMENT
A constant treatment volume of 5 ml/kg body weight was administered in all groups. 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.

FORMULATION
The test item was formulated in the vehicle (distilled water) in concentrations of 16.6, 50 and 150 mg/ml calculated by the active ingredient content. Formulations were prepared beforehand not longer than for three days and stored at 5 ± 3 °C before the administration.
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.
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 as defined by OECD 422). Sperm positive females were caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 5 or 10 ml 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 98 and 105 % in comparison to the nominal values.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.
Recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 98 % at ca. 1 mg/ml and 100 % at ca. 200 mg/ml).
A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical study. The substance proved to be stable in the vehicle in a refrigerator (at 5 ± 3 °C) for three days.
Duration of treatment / exposure:
42-59 days treatment/observation period (depending on the effectiveness of mating)
Frequency of treatment:
Daily, 7 days/week
Details on study schedule:
The experimental period involved 27 days of acclimatization (including 14 days for examination of estrous cycle) and 42-59 days treatment/observation period (depending on the effectiveness of mating) and necropsy days.
The day of first treatment is considered as Day 0 of examination.
Doses / concentrationsopen allclose all
Dose / conc.:
83 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
No. of animals per sex per dose:
12 males and 12 females per group
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.
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. Body weight was measured on day of necropsy for female animals subjected to organ weighing (selected for further examinations).

FOOD CONSUMPTION
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 (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 13 for female animals).

SERUM THYROID HORMONES
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 anesthesia.
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH).
Blood samples for determination of serum levels of thyroid hormones (T4 and TSH) were collected from animals as follows: from all dams and 2-7 pups per litter on post-partal/post-natal day 13; from all parent male animals at termination on Day 42.
Parameters measured: Thyroxine – free Tetra-iodothyronine and Thyroid-stimulating hormone.

HAEMATOLOGY
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 anesthesia.
Blood samples for hematology measurements were collected in tubes containing K3EDTA and tubes were filled up to the final volume (marked on the tubes). Blood were stored at 2-8 °C until analysis by Siemens ADVIA120.
Parameters measured: White Blood Cell (leukocyte) count, Red Blood Cell (erythrocyte) count, Hemoglobin concentration, Hematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Hemoglobin, Mean Corpuscular (erythrocyte) Hemoglobin Concentration, Platelet (thrombocyte) count, Reticulocytes, Differential† white blood cell count.

BLOOD COAGULATION
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 anesthesia.
Blood samples for determination of blood clotting times (APTT and PT) were collected in tubes containing 9NC Coagulation 3.2 %. Tubes were filled up to the final volume (marked on the tubes). Blood were centrifuged at 2500 rpm for 15 minutes within 20-30 minutes after the sampling. Supernatant plasma samples were stored at 2-8 °C and measured.
Parameters measured: Activated partial Thromboplastin Time, Prothrombin Time.

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 anesthesia.
Blood samples collected for clinical chemistry measurements were drawn in tubes Vacuette 2.5 ml Z Serum Sep C/A. At least 1.0 ml blood was collected into clinical chemistry tubes. Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. Serum samples were stored at 2-8 °C and measured.
Parameters measured: Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Sodium concentration, Potassium concentration, Albumin concentration, Total Protein concentration.

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 OF THE OFFSPRING
Each litter were 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.
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore, 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.
The number of nipples/areolae in male pups was counted on postnatal day 13.

SERUM THYROID HORMONES
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 anesthesia.
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH).
Blood samples for determination of serum levels of thyroid hormones (T4 and TSH) were collected from 2-9 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter).
Parameters measured: Thyroxine – free Tetra-iodothyronine and Thyroid-stimulating hormone.
Postmortem examinations (parental animals):
SACRIFICE
All animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® (details are presented in paragraph “Characteristics of anesthetics”) and were subjected to gross necropsy as follows: parental male animals after the optionally extended post-mating period on Day 42; dams on post-partum day 13 or shortly thereafter (Days 51, 54, 55, 57, 58 or 59).

GROSS NECROPSY
Gross necropsy was performed on each animal.
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, 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 for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.

ORGAN WEIGHTS
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.
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, 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.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (750 mg/kg bw/day).
In addition, kidneys of three male animals (no. 306, 311, 312 in the mid dose group), and uterus of three female animals (no. 229 in the low dose group, no. 322, 330 in the mid dose group) was processed and examined due to macroscopic findings (pyelectasia in the kidneys, hydrometra in the uterus).
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® (details are presented in paragraph “Characteristics of anesthetics”) and were subjected to gross necropsy in offspring on postnatal day 13 or shortly thereafter.

GROSS NECROPSY
Gross necropsy was performed on each animal.
Thyroid gland was preserved from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.
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, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs in any group, i.e. the parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 83, 250 or 750 mg/kg bw/day at the daily or at the detailed weekly clinical observations.
Noisy breathing was noted for one male animal at 750 mg/kg bw/day (1/12) on Day 28. Piloerection and slightly decreased activity were observed in one male animal at 750 mg/kg bw/day (1/12) between Days 34-36 and 34-41, respectively. Signs of bites around the vaginal orifice were noted for one control female animal (1/12) between gestation days 0 and 6.
These observations were considered to be individual signs and some of these were detected at the weekly detailed clinical observations, too (piloerection: 1/12 on Day 34; decreased activity: 1/12 on Days 34 and 41).
Mortality:
no mortality observed
Description (incidence):
There was no mortality at 83, 250 or 750 mg/kg bw/day groups during the course of study (male and female).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight development was undisturbed in male and female animals at 83, 250 or 750 mg/kg bw/day during the entire treatment period.
Statistically significant difference with respect to their control was detected at the higher mean body weight gain in male animals at 83 mg/kg bw/day between Days 13 and 20 and at 250 mg/kg bw/day between Days 34 and 41.
The mean body weight gain was lower than in the control group in male animals at 750 mg/kg bw/day during the entire observation period reaching statistical significance between Days 20 and 27 and for the study overall (between Days 0 and 41).
These slight changes in the body weight gain did not result in significant changes in the body weight of male animals in these groups, therefore were considered to be toxicologically not relevant.
In female animals at 250 mg/kg bw/day, statistically significantly higher mean body weight was detected when compared to the control on lactation day 0. This slight and transient change in the mean body weight was considered to be indicative of biological variation without toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes in the mean daily food consumption of male or female animals at 83, 250 or 750 mg/kg bw/day.
Statistical significance with respect to the control was detected in male animals administered with 750 mg/kg bw/day at the slightly lower mean daily food consumption during the first week of the treatment period.
The mean daily food consumption was comparable in the control and test item treated male animals at 83, 250 or 750 mg/kg bw/day during the post mating period and in female animals at 83, 250 or 750 mg/kg bw/day during the course of the pre-mating, gestation and lactation periods.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 83, 250 or 750 mg/kg bw/day.
The examined hematological parameters were comparable in male and female animals in the control and 83 and 750 mg/kg bw/day groups.
In male animals at 250 mg/kg bw/day, statistical significance was noted for the slightly lower red blood cell (erythrocyte) count (RBC), mean concentration of hemoglobin (HGB) and mean hematocrit value (HCT); in female animals at 250 mg/kg bw/day, statistical significance was noted for the slightly lower mean prothrombin time (PT).
All these slight changes were considered to have no toxicological relevance in the lack of dose dependence and due to the minor degree.
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 750 mg/kg bw/day (male or female).
Statistical significance with respect to the control was observed at the lower mean activity of aspartate aminotransferase (AST) in male animals at 83 and 250 mg/kg bw/day. Although the mean group value was comparable to the control, the activity of aspartate aminotransferase was slightly elevated in one male animal (1/5) at 750 mg/kg bw/day, however there were no related changes in the organ weight, necropsy or histopathology findings. Therefore, slight change in the AST of this animal was considered to be individual one
The mean concentration of creatinine (CREA) slightly exceeded the control value in male animals at 750 mg/kg bw/day group..
These statistically significant differences in male animals with respect to their controls were considered to have no toxicological importance because all values – mean and individual – remained within the historical control ranges (CREA, AST) or there was no dose relevance (AST). Moreover, there were no histological changes in related organs.
The examined clinical chemistry parameters were similar in female animals of the control and 83, 250 and 750 mg/kg bw/day groups.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation battery did not demonstrate any alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (selected male and female, control, 83, 250 or 750 mg/kg bw/day groups, on Day 42).
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.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 750 mg/kg bw/day).
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at the highest dose (750 mg/kg bw/day).
In the male animals belonging to the treated and control groups (12/12 at 750 mg/kg bw/day; 12/12 control), 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 at 750 mg/kg bw/day; 12/12 control), 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 four dams as follows: 1/12 control; 1/1 at 83 mg/kg bw/day; 2/2 at 250 mg/kg bw/day. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 control male; 1/5 male and 1/5 female at 750 mg/kg bw/day), acute hemorrhage in the lungs (1/5 male and 1/5 female in the control group; 1/5 male at 750 mg/kg bw/day) and thymic hemorrhage (1/5 male at 750 mg/kg bw/day) were detected sporadically. The pulmonary emphysema and hemorrhages in the lungs and thymus were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (1/5 male and 1/5 female in the control group; 1/5 male at 750 mg/kg bw/day) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Slight pyelectasia (one side or both sides) was observed in three male animals (3/3 at 250 mg/kg bw/day and 1/12 at 750 mg/kg bw/day). This finding without degeneration, inflammation or fibrosis is considered as slight individual lesion without pathological significance.
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 respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the eyes, the integumentary system, the male and female reproductive system or the central, or peripheral nervous system in the animals.
The cyto-morphology of endocrine glands was the same in the control and high dose treated animals.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
SERUM THYROID HORMONES
There were no toxicologically significant differences with respect to the control in the TSH thyroid hormone levels in parental male animals or in offspring sampled on postnatal day 13 at any dose levels.
Statistically significant difference with respect to the control was noted for the lower thyroid hormone (free T4) level in parental male animal at 750 mg/kg bw/day. The individual values were within or marginal to the historical control range (2.72 ± 0.39 ng/dl; n = 96; min = 1.91 ng/dl; max = 3.86 ng/dl), therefore this difference was considered to be toxicologically not relevant. Moreover, the TSH levels revealed no differences when compared to the control and there were no findings in regards to histopathological examinations of respective organs, there is no indication that hypothalamic–pituitary–thyroid axis was affected by the test item.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
A test item influence on the estrous cycle was not detected at any dose level (83, 250 or 750 mg/kg bw/day).
There were no statistically or biologically significant differences between the control and test item treated groups (83, 250 and 750 mg/kg bw/day) in the number or percentage of animals with regular or irregular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus, in the number or percentage of animals in prolonged estrous or diestrous during the pre-experimental and pre-mating periods.
Reproductive performance:
no effects observed
Description (incidence and severity):
The examined parameters of reproductive performance were not affected by the treatment with the test item at 83, 250 or 750 mg/kg bw/day in male or female animals.
There were no differences between the control and test item treated groups in copulatory and fertility indices (male and female animals) or in the pre-coital interval, conceiving days and gestation indices (female animals).

Details on results (P0)

DELIVERY OF DAMS - no effects observed
There were no toxicologically relevant differences in the evaluated parameters of delivery between the control and test item treated groups (83, 250 or 750 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 postpartal day 0, in the percentage of dams with viable offspring on postpartal day 0 or in the live birth index.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: reproductive toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
The percentage of offspring with signs (cold and not suckled) at 83, 250 and 750 mg/kg bw/day was higher than in the control group on postnatal day 0. However, these signs were considered to be toxicologically not relevant in the lack of dose relevance and the signs were transient – detected only shortly after the delivery – and were not associated with depression on the development of the offspring.
Occasionally, other clinical signs were also observed: pale (1 % in the control and 1 % at 83 mg/kg bw/day), abnormal tail position (1 % in the control), smaller than normal pup (1 % in the control), hemorrhage on the nose (1 % at 83 mg/kg bw/day), cyanotic skin (1 % at 750 mg/kg bw/day), uncleaned pup (11 % at 750 mg/kg bw/day) which however were considered to have no toxicological relevance.
Mortality / viability:
no mortality observed
Description (incidence and severity):
The offspring’s extra uterine mortality was not affected by the test item.
There were no significant differences between the control and test item treated groups (83, 250 or 750 mg/kg bw/day) in the mean number of dead offspring (including missing pups) per litter.
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 750 mg/kg bw/day) groups in the survival indices.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight development of the offspring was unaffected by the test item.
The mean litter weight and mean pup weight as well as the mean litter weight gain and mean pup weight gain were comparable in the control and in all test item treated groups (83, 250 and 750 mg/kg bw/day) on postnatal days 0, 4 and 13.
Considering the offspring’s body weight in males and females separately, statistically significant difference with respect to the control was detected at the slightly lower mean weight of male pups at 250 and 750 mg/kg bw/day and female pups at 750 mg/kg bw/day on postnatal day 4. The difference with respect to the control was with minor degree and it was considered to be toxicologically not relevant.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
The anogenital distances (absolute and normalized in male or female offspring) or nipple retention (male) were not adversely affected by the test item treatment at 83, 250 and 750 mg/kg bw/day.
At 83 mg/kg bw/day, statistical significance was observed for the slightly shorter mean normalized anogenital distance in female pups.
At 750 mg/kg bw/day, statistically significantly shorter mean absolute and normalized anogenital distance was noted for male and female pups as well.
These differences in the high dose group with respect to the control are in good correlation with the slightly lower mean pup weight in the high dose group, therefore were not considered to be toxicologically relevant.
Nipples/areoles were not visible in any of the examined male offspring in the control or 83, 250 or 750 mg/kg bw/day groups on postnatal day 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
There were no macroscopic changes in the organs or tissues of the two stillborn offspring (1/1 in control and 1/1 at 83 mg/kg bw/day) subjected to necropsy on the day of delivery. The umbilical cord was connected to the control stillborn.
Other effects:
no effects observed
Description (incidence and severity):
SEX DISTRIBUTION
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.

SERUM THYROID HORMONES
There were no toxicologically significant differences with respect to the control in the TSH thyroid hormone levels in offspring sampled on postnatal day 13 at any dose levels.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL (P0) (rat M/F) ≥ 750 mg/kg bw/day, based on reproductive performance
NOAEL (F1) (rat) ≥ 750 mg/kg bw/day, based on developmental toxicity
Executive summary:

The toxic potential of test item to cause effects on reproduction and development was investigated performing a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, according to the testing guideline OECD 422.

The substance was administered orally (by gavage) to rats at doses of 83, 250 and 750 mg/kg bw/day by active ingredient (corresponding to uncorrected dose of 92.7, 279.3 and 838 mg/kg bw/day, respectively). The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study and the concentrations in the dosing formulations were found to varie within the range of 98 and 105 % in comparison to the nominal values, 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). Females were additionally exposed through the gestation period and up to lactation days 13-15, i.e. up to the day before necropsy (altogether for 51-59 days). The dams were allowed to litter and rear their offspring up to day 13 post-partum.

No deaths occurred during the course of study (male and female). No signs of systemic toxicity were detected at any dose level in parent animals.

A test item influence on the estrous cycle was not found at any dose level. No 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 were recorded.

There were no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose (parental male or 13-day offspring).

No test item effect on the mortality, clinical signs, body weight development or necropsy findings were detected in the offspring. The anogenital distance (male and female) or nipple retention (male) were not affected.

Conclusion

Under the conditions of the study, the test item did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female.

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

NOAEL for systemic toxicity of male/female rats ≥ 750 mg/kg bw/day

NOAEL (P0) (rat M/F) ≥ 750 mg/kg bw/day, based on reproductive performance

NOAEL (F1) (rat) ≥ 750 mg/kg bw/day