Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 28 to May 22, 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 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop ZRT, Budapest.
- 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: 90 - 95 days.
- Weight at study initiation: 326 – 435 g for male animals; 208 – 265 g for female animals. The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: before mating, 2 animals of the same sex/cage; during mating 1 male and 1 female per cage; pregnant females were individually housed; males after mating were housed 2 animals per cage. Type III polypropylene/polycarbonate cages (22 × 32 × 19 cm).
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, Germany , ad libitum.
- Water: tap water, as for human consumption, ad libitum.
- Acclimation period: 48 days.

FOOD AND WATER CERTIFICATES
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:
water
Details on exposure:
FORMULATION
Test item was formulated in the vehicle (distilled water) in concentrations of ca. 15, 45 and 151 mg/ml by the active ingredient content. Formulations were prepared in the formulation laboratory of the Test Facility in a frequency based on the stability feature of the test item in the vehicle (not longer than for three days beforehand and stored at 5 ± 3 °C until use).
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. For one female animal in each of the mid and high doses, male pair was replaced by a proven male.
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 421). 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 20 ml of each formulation and five aliquots of 20 ml control substance (vehicle) were taken and analyzed.
Concentration of the test item in the dosing formulations varied in the range of 103 and 108 % of the nominal values at both analytical occasions.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. Recovery of the test item from water at 1 and 200 mg/ml concentrations in water was 99 and 96 % of nominal concentrations, respectively.
The test item was stable in water at 1 and 200 mg/ml concentrations at room temperature for one day (101 and 107 % of nominal concentrations, respectively) and in a refrigerator (5 ± 3 °C) for three days (105 and 108 % of nominal concentrations, respectively).
Duration of treatment / exposure:
Males: during the pre-mating, mating and post-mating periods, i.e. 48 days.
Females: during the pre-mating, mating, gestation and lactation periods, i.e. 48 – 55 days.
Frequency of treatment:
Daily up to the day before the necropsy, 7 days per week.
Details on study schedule:
Acclimatization: 48 days, including 14 days for examination of estrous cycle.
Pre-mating: 14 days.
Mating: 1-21 days.
Post-mating for males: 13-33 days.
Gestation for females: 22 days (for not mated and non-pregnant female animals necropsy was conducted on Day 48 or 51).
Lactation for females and pups: 14-17 days.
Doses / concentrationsopen allclose all
Dose / conc.:
75.6 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient, corresponding to an uncorrected dose of 100 mg/kg bw/day
Dose / conc.:
226.8 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient, corresponding to an uncorrected dose of 300 mg/kg bw/day
Dose / conc.:
756 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient, corresponding to an uncorrected dose of 1000 mg/kg bw/day
No. of animals per sex per dose:
12 animals per group and sex
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, considering the peak period of anticipated effects after dosing.
More 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.

BODY WEIGHT
All parental animals were weighed 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 data were reported individually for adult animals. Individual body weight change was calculated.

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, 41 and 48 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

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

BIOCHEMISTRY - THYROID HORMONES
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH) from all parent male animals at termination on Day 48.
Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. All samples for T4 and TSH determination were stored between minus 15 and minus 20 °C until measurement.
Blood samples from the day 13 the adult males were assessed for serum levels of thyroid hormones (T4 and TSH).
The quantitative determination of thyroid hormones (T4 and TSH) in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment started from each animal was being considered for study for two weeks. Animals exhibited typical 4-5 days cycles were included in the study preferably. 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 smear also was prepared on the day of the necropsy.
Each morning a vaginal smear were prepared and stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope.
Litter observations:
DELIVERY/GESTATION OBSERVATION
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations and any evidence of abnormal deliveries were recorded. The duration of gestation was recorded and is calculated from day 0 of pregnancy.

DAMS OBSERVATION
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 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).
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.

LITTER OBSERVATION
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.
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.

ANOGENITAL DISTANCE
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight.

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.

NIPPLES/AREOLAE
The number of nipples/areolae in male pups was counted on postnatal day 13.

BIOCHEMISTRY - THYROID HORMONES
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH):
- from at least two pups per litter on post-natal day 4 (if it was feasible) - pup blood was pooled by litter.
- from all dams and at least two pups per litter at termination on day 13
Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. All samples for T4 and TSH determination were stored between -15 and -20 °C until measurement.
Blood samples from the day 13 pups were assessed for serum levels of thyroid hormones (T4 and TSH). Further assessment of T4 and TSH in blood samples from the dams and day 4 pups were not performed because there were no relevant changes in the examined samples (based on observations in postnatal day 13 offspring and parental male animals).
The quantitative determination of thyroid hormones (T4 and TSH) in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.
Postmortem examinations (parental animals):
GROSS PATHOLOGY
Gross necropsy was performed on each animal. Terminally (one day after the last treatment), animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® (details are presented in paragraph “Characteristics of anesthetics”)
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 uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries 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 pharynx.

Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.
All organs showing macroscopic lesions were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above).
Selected organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved as described above.

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 adult male animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported. The thyroid weight will be determined – if relevant – after fixation.
Paired organs were weighed together.

HISTOPATHOLOGY
Detailed histological examinations were performed on the ovaries, testes, epididymides – with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure – in the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
These organs were also processed and examined histologically in not mated and non-pregnant females and males these females cohabited with in the low and middle dose groups.
In addition, the following organs were processed and examined due to the macroscopic findings:
- right epididymis of one male in the low dose group,
- kidney of one male and one female in the low dose group, and three males (302, 312 and 401, pyelectasia) in the mid and high dose groups,
- uterus of three dams in the low and mid doses,
- seminal vesicles with coagulating gland of one male in the high dose,
- stomach of three males and one female in the high dose.
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):
GROSS PATHOLOGY
Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities. All organs showing macroscopic lesions were preserved in 4 % buffered formaldehyde solution (except testes and epididymides).
Selected organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved.
Gross necropsy was performed on each animal. Terminally (one day after the last treatment), animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® (details are presented in paragraph “Characteristics of anesthetics”)
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 uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries 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 one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with pharynx.

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 adult male animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported. The thyroid weight will be determined – if relevant – after fixation.
Paired organs were weighed together.

HISTOPATHOLOGY
Detailed histological examinations were performed on the ovaries, testes, epididymides – with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure – in the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
These organs were also processed and examined histologically in not mated and non-pregnant females and males these females cohabited with in the low and middle dose groups.
In addition, the following organs were processed and examined due to the macroscopic findings:
- right epididymis of one male in the low dose group,
- kidney of one male and one female in the low dose group, and three males (302, 312 and 401, pyelectasia) in the mid and high dose groups,
- uterus of three dams in the low and mid doses,
- seminal vesicles with coagulating gland of one male in the high dose,
- stomach of three males and one female in the high dose.
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.
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/ pre-natal mortality (intrauterine mortality)
- Post-natal mortality /Extra-uterine mortality
- Survival Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In male animals, piloerection and wet bedding material were detected at 756 mg/kg bw/day dose levels at the daily and at the detailed weekly clinical observations; at 226.8 mg/kg bw/day decreased activity and decreased body tone were observed in one male at the daily and at the detailed weekly clinical observations; at 75 mg/kg bw/day no clinical sign was detected at the daily and at the detailed weekly clinical observations.
In female animals, piloerection, decreased body tone (during gestation) and wet bedding material were detected at 756 mg/kg bw/day dose levels at the daily and at the detailed weekly clinical observations; at 226.8 mg/kg bw/day piloerection, decreased activity and decreased body tone were observed in one female daily and at the detailed weekly clinical observations; at 75 mg/kg bw/day no clinical sign was detected at the daily and at the detailed weekly clinical observations.

The stool was soft and red in each cages of male and female animals of 75.6, 226.8 or 756 mg/kg bw/day groups from Day 1 up to the termination of the treatment.

Daily clinical observations
The behavior and physical condition of animals were normal in male and female animals in the control and at 75.6 mg/kg bw/day.
At 226.8 mg/kg bw/day, decreased activity on Days 39-47, stool around the anus on Days 42-47, decreased body tone on Days 43-47 was observed in one male (1/12) animal.
Piloerection on Days 23-42 and decreased body tone on Days 23-44 were detected for one not mated female animal (1/12, not mated) at this dose.
At 756 mg/kg bw/day in male animals, decreased activity on Days 42-47 (1/12), decreased body tone on Days 40-47 (1/12), piloerection from Day 24 or 40 to Day 27 or 40 or 41 or 47 (5/12), hunched back on Days 46-47 (1/12), noisy breathing on Days 37-41 (1/12) and wet bedding material on Days 7-47 (12/12) were observed.
In female animals at 756 mg/kg bw/day, piloerection (4/12, including one not mated and two non-pregnant females) and decreased body tone (3/12, including one not mated and two non-pregnant females), stool around the anus (1/12, one not mated female) and wet bedding material (12/12 in the pre-mating period and 1/1 in the post-mating period of one not mated female) was observed.
Soft, red stool caused by the test item was detected in the bedding material in each cage: male and female animals of 75.6, 226.8 or 756 mg/kg bw/day groups from Day 1 up to the termination of the treatment. Red color of stools was indicative of presence of the test item or its metabolite in the intestinal tract.

Detailed weekly clinical observations
The behavior and physical condition of animals were considered to be normal in male and female animals in the control and at 75.6 mg/kg bw/day during the detailed weekly clinical observations.
At 226.8 mg/kg bw/day, decreased activity, decreased body tone and stool around the anus for one male animal and piloerection and decreased body tone for one not mated female animal – as described above – were also detected at the detailed weekly observation.
In male animals at 756 mg/kg bw/day, piloerection (3/12), decreased body tone (2/12) and noisy breathing (1/12) – as described above – were also detected at the detailed weekly observation. In females at this dose, similarly to the daily observations, piloerection and weak body tone were observed for one not mated and for two non-pregnant females.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in all the 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 and female animals at 75.6, 226.8 and 756 mg/kg bw/day. However, the difference with respect to the control in the mean body weight was low (< 10 %) in male animals at 75.6 mg/kg bw/day and was not related to doses in female animals at 75.6 and 226.8 mg/kg bw/day. Thus, the toxicological relevance of these findings at 75.6 mg/kg bw/day (male and female) and 226.8 mg/kg bw/day (male) is questionable.
The mean body weight was reduced in male animals at 75.6, 226.8 and 756 mg/kg bw/day in a dose related manner during the course of the entire study attaining statistical significance at 226.8 and 756 mg/kg bw/day on Days 27, 34, 41 and 48. The mean body weight gain was statistically significantly lower in all the test item treated groups on week 1, in the low and mid doses on week 2, in the mid and high doses on week 4 and in the mid dose on week 7. Statistically significantly reduced body weight gain was also observed in all the test item treated groups in the entire treatment period (between Day 0 and 48).
In females at 75.6, 226.8 and 756 mg/kg bw/day statistically significantly reduced mean body weight was observed during the entire study (except gestation day 0 in the mid dose where the decrease was not statistically significant). The body weight gain was statistically significantly lower than in the control group on the first week of treatment and in the whole pre-mating period (weeks 1 and 2) at 75.6, 226.8 and 756 mg/kg bw/day, on gestation weeks 2 and 3 at 75.6 mg/kg bw/day, on gestation weeks 1 and 2 at 226.8 mg/kg bw/day, on gestation weeks 1 and 3 at 756 mg/kg bw/day and during the whole gestation period (gestation weeks 1-3) in all the test item treated groups. In the whole lactation period (between lactation days 0 and 13) of female animals statistically significantly higher mean body weight gain was observed at 75.6 and 226.8 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse changes in the food consumption of male and female animals at any dose level (75.6, 226.8 and 756 mg/kg bw/day). Slightly lower mean daily food consumption of female animals in each test item treated group with respect to the control was not related to doses on pre-mating week 1 and on gestation weeks 1 and 3. Therefore these findings were considered to be toxicologically not relevant.
Statistically or biologically significant differences were not seen in the mean daily food consumption of male animals at any dose level with respect to control group during the entire observation period (pre-mating and post mating period).

However, on the first week of treatment in all of the test item treated groups slightly reduced daily mean food consumption was noted in males. This change was without statistical significance moreover it was transient, therefore was considered to have no or little toxicological relevance.
In female animals, the mean daily food consumption was statistically significantly less than in the control group at 75.6, 226.8 and 756 mg/kg bw/day in the first week of the pre-mating period, at 75.6 and 756 mg/kg bw/day in the first and third week of the gestation period. In the lactation period, statistically significantly higher mean daily food consumption was noted at 226.8 mg/kg bw/day. These occasionally reduced food consumption data of female animals without clear dose relevance are in good correlation with the mean body weight of the female animals. Thus, these changes are considered to have little or no toxicological meaning.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum thyroid hormone levels (T4 and TSH) were not affected in parental male animals or in PN13 offspring at 75.6, 226.8 and 756 mg/kg bw/day.
There were no significant differences with respect to the control in the TSH thyroid hormone levels in parental male animals or in the T4 thyroid hormone levels in offspring sampled on postnatal day 13 at any dose levels.
Statistically significant difference with respect to the control was noted for the lower mean thyroid hormone (free T4) level in parental male animals at 75.6, 226.8 and 756 mg/kg bw/day without clear dose response. Additionally, the mean values were within the historical control range. Thus, these differences were judged to be toxicologically not relevant.
Statistically significantly higher mean TSH thyroid hormone level was observed in PN13 offspring at 756 mg/kg bw/day. One pup had highly elevated value of TSH resulting in a higher mean value. This difference was considered to have no or little toxicological meaning.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examinations did not reveal any test item related alterations in the selected organs or tissues of male and female animals (testes, epididymides, ovaries) at the highest dose (756 mg/kg/bw/day).
Test item related focal squamous cell hyperplasia in the mucous membrane of non-glandular stomach was observed in three male and one female animal at 756 mg/kg bw/day.
In the male animals the investigated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides was normal in all cases. Decreased amount of secrete in the seminal vesicle on one side at 756 mg/kg/bw/day (no. 406) occurred. This lesion, without inflammatory or degenerative lesions could be considered as individual disease.
In addition, sperm granuloma (one side) in the epididymis of one male animal at 75.6 mg/kg/bw/day was observed. This phenomenon could be considered as individual disorder without toxicological significance.
In the female animals the ovaries 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 female animal in the control (1/11 dam), in two females at 75.6 mg/kg bw/day (2/2 dams), in one female at 226.8 mg/kg bw/day (1/1 dam) and in one female at 756 mg/kg bw/day (1/9 dam). This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and could be in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
Histological examination revealed focal squamous cell hyperplasia in the mucous membrane of non-glandular stomach in three male and one female animal at 756 mg/kg bw/day (no. 405, 406, 411, 422). This lesion was not accompanied with marked infiltration of inflammatory cells in the lamina propria or ulceration.
The squamous cell hyperplasia in the mucous membrane of non-glandular stomach occurred only in the high dose group and could be in possible connection with the local effect of high dose of test item and could be considered as a slight, reversible lesion.
Renal pyelectasia was observed in some male animals at 75.6 mg/kg bw/day (1/1), at 226.8 mg/kg bw/day (2/2) and at 756 mg/kg bw/day (1/1) in accordance with necropsy findings of these animals. Pyelectasia without degenerative, inflammatory or other histopathological lesions is a common finding in experimental rats of this strain and has no toxicological relevance in this study.

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 (75.6, 226.8 and 756 mg/kg bw/day).
There were no statistically or biologically significant differences between the control and treated groups (75.6, 226.8 and 756 mg/kg bw/day) in 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 during the pre-experimental and pre-mating periods. The number or percentage of animals in prolonged diestrous was slightly higher than the control at 226.8 and 756 mg/kg bw/day during the pre-mating period.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The fertility index of male and female animals in the high dose group (82 %) was slightly lower than in the control group (92 %), however this slight difference was considered to be indicative of biological variation and not related to the test item.
Statistically significant differences between the control and test item treated groups were detected in male and female animals at 756 mg/kg bw/day at the lower fertility indices as two out of eleven sperm positive females were not pregnant.
Statistical significance was also observed at the higher fertility indices at 75.6 and 226.8 mg/kg bw/day in male and female animals as one pair of control group mated without pregnancy resulting in slightly lower fertility indices. This finding was not considered as biologically relevant.
Statistical significance was detected at the slightly lower copulatory index in male and female animals at 226.8 and 756 mg/kg bw/day comparing to their control. One pair in each group failed mating. Considering the minor degree of these changes, these differences with respect to their control were not considered to be toxicologically relevant.

Details on results (P0)

There were no relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (75.6, 226.8 or 756 mg/kg bw/day).
Statistically significant difference was observed at the slightly shorter pregnancy at 756 mg/kg bw/day when compared to their control. This slight difference was considered to have no toxicological relevance.
The mean number of implantation sites, total birth, pregnant, liveborns and alive pups on post-natal day 0 were slightly lower than in the control group in dams at 756 mg/kg bw/day without statistical significance. However, all values were well within the historical control ranges therefore these slight differences have no toxicological relevance.
The mean number of stillborns, pre- or post-natal loss, and the livebirth index were comparable in all groups and the differences between the control and test item treated doses did not obtain any statistical significance.

Effect levels (P0)

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Dose descriptor:
NOAEL
Effect level:
75.6 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
LOAEL
Effect level:
226.8 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
756 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

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 756 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 as 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 cyanotic skin (1% in the control) which however were considered to+ have no toxicological relevance.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
There were no significant differences between the control and test item treated groups (75.6, 226.8 or 756 mg/kg bw/day) in the mean number of dead offspring (including missing pups) per litter.
There were no significant differences between the control and test item treated (75.6, 226.8 or 756 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 was reduced in the offspring at 756 mg/kg bw/day. Maternal toxicity at 756 mg/kg bw/day could induce this kind of body weight change of pups.
The mean litter weight, body weight, litter weight gain and body weight gain of offspring was also lower than in the control group at 75.6 and 226.8 mg/kg bw/day. These differences with respect to the control were with minor degree and not related to doses therefore were considered to have little or no toxicological relevance.
The mean litter weights and mean pup weights as well as the mean litter weight gain and mean pup weight gain were lower than in the control in all test item treated groups (75.6, 226.8 or 756 mg/kg bw/day) on postnatal days 0, 4 and 13 attaining statistical significance in most of the cases.
The mean pup weight gain between postnatal days 0 and 13 was -14.1 % in the low dose, -12.3 % in the mid dose and -26.8 % in the high dose groups.
The offspring’s mean body weight in males and females separately was also statistically significantly reduced at 75.6, 226.8 or 756 mg/kg bw/day in the most of the cases.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
The stomach was filled up with gas in one dead pup (1/1) in the control on post-natal day 1.
Autolyzed visceral organs and empty stomach were detected in one dead pup (1/2) at 75.6 mg/kg bw/day on post-natal day 1. Yellow foamy content in the stomach was observed in one dead pup (1/2) in this groups on post-natal day 2.
There were no macroscopic changes (milk in the stomach) in the organs or tissues of the dead pup (1/1) at 226.8 mg/kg bw/day on post-natal day 3.
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.

ANOGENITAL DISTANCE AND NIPPLE RETENTION
The anogenital distances were reduced in male and female offspring at 756 mg/kg bw/day. The differences with respect to the control at 75.6 and 266.8 mg/kg bw/day were with minor degree and were not related to doses therefore were considered to have no toxicological relevance; this can be a consequence of maternal toxicity.
The nipple retention (male) were not affected by the test item treatment at 75.6, 266.8 or 756 mg/kg bw/day.
Statistical significances were observed at the shorter absolute anogenital distances in male pups of the 75.6, 226.8 and 756 mg/kg bw/day treated dams and at the shorter anogenital distances (absolute and normalized as well) in female pups at 75.6 and 756 mg/kg bw/day. The normalized anogenital distance of male pups at 756 mg/kg bw/day was slightly longer than in the control group.
Nipples/areoles were not visible in any of the examined male offspring in the control or 75.6, 226.8 or 756 mg/kg bw/day groups on postnatal day 13.

Effect levels (F1)

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Dose descriptor:
NOAEL
Generation:
F1
Effect level:
226.8 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
756 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: due to 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 for systemic toxicity (male and female): 75.6 mg/kg bw/day
NOAEL for reproductive performance (male and female): 756 mg/kg bw/day
NOAEL for F1 offspring: 226.8 mg/kg bw/day
LOAEL for F1 offspring: 756 mg/kg bw/day due to maternal toxicity
Executive summary:

The toxic potential of test item and its possible effects on reproduction and development upon repeated oral dosing (by gavage) of rats at 75.6, 226.8 or 756 mg a.i./kg bw/day (equivalent to 100, 300 and 1000 mg/kg bw/day) compared to control animals was assessed. Four groups of Han:WIST rats consisting of 12 animals per group and sex were dosed once daily. 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 animals was checked two times during the study and the test item concentrations in the dosing formulations varied in the acceptable range between 103 and 108 % of the nominal values.

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 48 days). Females were additionally exposed through the gestation period and up to lactation days 13-16, i.e. up to the day before necropsy (altogether for 51-55 days).

The dams were allowed to litter and rear their offspring up to day 13 post-partum.

All parental animals were subjected to gross pathology one day after the last treatment.

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

Clinical signs of systemic toxicity related to the test item were detected at 226.8 (decreased activity, decreased body tone, diarrhea in single animals) and 756 mg/kg bw/day (decreased activity, decreased body tone, piloerection, hunched back, noisy breathing with low incidence and wet bedding material) in male and female animals at the daily or at the detailed weekly clinical observations.

The stool was soft and red in each animal at 75.6, 226.8 and 756 mg/kg bw/day.

The body weight gain and body weight were reduced in male and female animals of all the groups. The difference with respect to the control in the mean body weight was low (< 10 %) in male animals at 75.6 mg/kg bw/day and was not related to doses in female animals at 75.6 and 226.8 mg/kg bw/day during the gestation and lactation period. Thus, the toxicological relevance of these findings at 75.6 mg/kg bw/day (male and female) and 226.8 mg/kg bw/day (female) is questionable.

The mean daily food consumption was not adversely affected by the test item in male or female animals at 75.6, 226.8 or 756 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods).

A test item influence on the estrous cycle was not detected at any dose level.

There were no toxicologically relevant differences between the control and test item treated groups in the evaluated delivery parameters of dams.

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

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).

Test item related changes in the stomach (thickened wall, erythematous mucous membrane) were observed in some male and female animals at 756 mg/kg bw/day. Presence of the test item or its metabolite(s) in the stomach and/or intestines were detected in several male (all dose levels) and female animals (226.8 and 756 mg/kg bw/day) at the gross macroscopic observation.

There were no test item related adverse changes in the organ weights (absolute and relative to body or brain weights) of brain, testes, epididymides and seminal vesicles of male animals at any dose level.

Histopathological examinations of the selected sexual organs (ovaries, testes, epididymides) did not reveal any test item related changes at any dose level. Focal squamous cell hyperplasia in the mucous membrane of non-glandular stomach was detected in three male animals and one female animal at 756 mg/kg bw/day. This lesion was in connection with the local effect of high dose of the test item and might be considered as a slight, reversible lesion.

Test item related reduced body weight development of the offspring was found at 756 mg/kg bw/day; such finding was due to the reduced body weight after repeated oral administration of dams from birth to post-natal day 13. Maternal toxicity could be assumed in development of body weight changes of offspring.

No adverse findings on the mortality, clinical signs or at necropsy were detected in the offspring terminated as scheduled.

Conclusion

NOAEL for systemic toxicity (male and female): 75.6 mg/kg bw/day

NOAEL for reproductive performance (male and female): 756 mg/kg bw/day

NOAEL for F1 Offspring: 226.8 mg/kg bw/day