Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the conditions of an OECD 422 compliant study, the test item caused signs of systemic toxicity in male and female Hsd.Han: Wistar rats at 1000 mg/kg bw/day administered by oral gavage. As a consequence an influence on the estrous cycle, delivery data of dams (higher pre-implantation loss, lower birth mean), reproductive performance of male and female animals (prolonged pre-coital and conceiving periods) were also observed at 1000 mg/kg bw/day. The development of the F1 offspring was also impaired at 1000 mg/kg bw/day due to poor general condition of the dams at this dose level: clinical signs with higher incidence, higher mortality and reduced body weight gain and body weight were observed with respect to their control.

Based on these observations the No Observed Adverse Effect Levels (NOAELs) were determined as follows:

NOAEL for systemic toxicity of male and female rats: 300 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 300 mg/kg bw/day

NOAEL for F1 Offspring: 300 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-31 to 2018-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 88-90 days old
- Weight at study initiation: Males: 346 – 396 g; Females: 203 – 237 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- 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 drinking water is periodically analyzed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 -70 %
- Air changes: > 10 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 20, 60 and 200 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility daily.
Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Concentration of the test item in the dosing formulations varied in the range of 96 and 100 % of the nominal values at both analytical occasions.

VEHICLE
- Justification for use and choice of vehicle: common vehicle according to the relevant guidelines
- Concentration in vehicle: 20, 60, 200 mg/mL
- Amount of vehicle: constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females were cohabited with the same male until copulation occurred or two weeks elapsed. Three female animals (no. 425, 428, 431) at the high dose failed to mate during the 14-days period, therefore mating period was prolonged and their partners were exchanged with each other.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and were measured on 2 occasions, during the first and last treatment weeks. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.

Date of sampling: September 12 and October 31 2017.
Date of analysis: September 13 and November 01 2017.

Concentration of the test item in the dosing formulations varied in the range of 96 and 100 % of the nominal values at both analytical occasions.
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 sunflower oil was 97 % and 100 % of nominal concentrations at ~ 10 mg/g and ~ 200 mg/g, respectively.

The test item was stable in sunflower oil in concentrations of ~10 mg/g and ~200 mg/g at least three days at room temperature and in refrigerator.
Duration of treatment / exposure:
Males: 51 days
Females: 51 to 71 days due to prolonged mating period
Frequency of treatment:
daily, 7 d per week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a previous dose range finder study
- Rationale for animal assignment: random based on body weight, and estrous cycle of female animals was also considered at the randomization.

Estrous cycle was evaluated and considered at randomization; however, several animals that failed to exhibit typical 4-5 days cycles were included in the study due to the large number of animals with irregular cycle
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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.
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: Yes
- Time schedule for examinations: first day of dosing, weekly thereafter and on the day of necropsy

FOOD CONSUMPTION: Yes
- Time schedule: weekly during treatment phase except during mating

FOOD EFFICIENCY: no

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last treatmend, i. e. day of necropsy
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight (approx. 16 h)
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in table 1 & 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after last treatmend, i. e. day of necropsy
- Animals fasted: Yes, overnight (approx. 16 h)
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in table 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks. 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 copulation.
Vaginal smear were also prepared on the day of the necropsy.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Please refer to table 4.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examinations were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma.
In addition, these organs were processed and examined histologically in not mated, non-pregnant or not delivered females and males these females cohabited with in the low and middle dose groups.

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals (n=5 animals/sex/group) in the control and high dose groups.

In addition, liver was also processed and examined histologically in all male animals due to necropsy findings observed in several male animals (pale and nutmeg-like pattern). Seminal vesicle with coagulating gland was also processed and examined for one male (no. 205) at 100 mg/kg bw/day due to the necropsy finding (smaller than normal, one side). Uterus of one female in the low dose group (no. 232) was processed and examined as well due to macroscopic finding (hydrometra).
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations macroscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGTHS: No
Statistics:
The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose were calculated. Results were evaluated in comparison with values of control group (i.e. control value).
Reproductive indices:
Parental Males:
- Percentage of pairings
- Percentage of fertile pairings
- Percentage of infertile males
- Male copulatory index: Number of males with confirmed mating / total number of males cohabited x 100
- Male fertility index: Number of males impregnating for a female / total number of males with confirmed mating x 100

Parental Females:
- Percentage of pairings / sperm positive females
- Percentage of pregnant females
- Percentage of sperm positive, but non-pregnant females
- Percentage of non-mated females
- Female copulatory index: Number of females with confirmed mating / total number of females cohabited x 100
- Female fertility index: Number of pregnant female / number of spermpositive females x 100
- Gestation index: Number of females with liveborn pubs / number of pregnant females x 100
- Duration of pregnancy (days)
- Number of implantations / dams
- Number of dams with live pups on lactation days 0, 4 and 13
- Post-implantation mortality: Number of implantations - Number of liveborns / Number of implantations x 100

Offspring viability indices:
Offspring:
Litter weight on postnatal days 0, 4 and 13
- Mean body weight gain per litter between postnatal days 0-4, 4-13 and for overall between post-natal days 0 and 13
- Number of live births per litter, and number of viable pups per litter on postnatal days 0, 4 and 13
- Survival Index of pups on postnatal day 13: Number of live pups on postnatal days 13 / Number of live borns x 100
- Sex ratio % (on postnatal days 0 and 13): Number of pups examined - Number of males (females) / Number of pups examined x 100
- Normalized anogenital distance
- Number of nipples/areolae in male pups
- T4 on post-partum day 13
- postnatal mortality: Number of liveborns - Number of live pups on PND 13 / Number of liveborns x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Daily Observations:
Test item related clinical signs were observed at 1000 mg/kg bw/day (male and female) and at 300 mg/kg bw/day (female) at the daily clinical observations: decreased activity, salivation, nuzzling up the bedding material or piloerection were observed with variable incidence from Day 6 onwards.
There were no clinical signs in control male animals (12/12), i.e. these parental animals exhibited normal behavior and physical condition with no abnormalities during the treatment period. Abnormal tail position was noted for one control female dam (1/11) from gestation day 0 up to the study termination. All other control female animals showed normal behavior and physical condition throughout the pre-mating, post-mating, gestation or lactation period.
At 100 mg/kg bw/day, exophthalmia of the right eye of was observed in one male animal (1/12) from Day 2 until the end of the study. There were no clinical signs in the other male animals (11/12) during the observation period or in female animals of this group (12/12) female during the pre-mating, post-mating, gestation or lactation periods.
At 300 mg/kg bw/day, all male animals were symptom-free during the entire observation period.
Female animals at 300 mg/kg bw/day showed salivation (2/12), nuzzling up the bedding material (2/12) during the pre-mating period as well as during the gestation period (2/10, both signs). There were no clinical signs in dams of this group (10/10) during the lactation period.
Piloerection was noted for one of the non-pregnant female animals at 300 mg/kg bw/day (1/2) from Day 38 up to the termination.
Salivation (6/12), nuzzling up the bedding material (6/12), piloerection (4/12) and decreased activity (2/12 males) were observed in male animals at 1000 mg/kg bw/day. These signs were observed during the pre-mating, gestation and lactation periods as well as in non-pregnant or not mated female animals at 1000 mg/kg bw/day as indicated in table 5.
Decreased body tone and increased respiratory rate were noted for one female in the high dose group only on lactation day 0 probably due to the delivery process.

Detailed Weekly Observations:
Test item related clinical signs (piloerection or decreased activity) were observed at 1000 mg/kg bw/day (male and female) and at 300 mg/kg bw/day (female) at the detailed weekly clinical observations. Additionally, decreased body tone and increased respiratory rate was noted for one female animal at 1000 mg/kg bw/day on the day of the delivery. In the male animals of control group, there were no clinical signs.
In one control female animal (1/11), abnormal tail position was detected also at the weekly observations from gestation day 0 up to the end of the study.
At 100 mg/kg bw/day, exophthalmia of the right eye of was noted for one male animal (1/12) from Day 7 onwards. Female animals of the low dose group showed normal physical condition and behavior at the detailed weekly clinical observations during the pre-mating, gestation and lactation periods.
At 300 mg/kg bw/day, all male animals were normal at each weekly clinical observation.
In the female group of 300 mg/kg bw/day, piloerection was noted for single non-pregnant animal (1/2) on study Days 41 and 48. All other animals were normal during the detailed weekly observations.
At 1000 mg/kg bw/day, piloerection and decreased activity was noted for male animals on Days 20 (2/12) and 27 (1/12). Piloerection was only observed in some male animal on Days 41 (2/12), 48 (1/12) and day 50 (1/12).
In the female animals at 1000 mg/kg bw/day, decreased activity (1/10 on gestation days 0, 7, 13 and 20; 1/10 on lactation day 0) and piloerection (1/10 on gestation days 0, 7, 13 and 20; 3/10 on lactation days 0 and 4; 1/10 on lactation day 13) were seen. One dam of the high dose group, showed also decreased body tone and increased respiratory rate at the detailed observation on the day of delivery (lactation day 0).

Functional observations:
Test item related piloerection was noted for one female in the 1000 mg/kg bw/day group (1/10) at the functional observations.

Functional observations did not demonstrate any test item related changes at 100, 300 mg/kg bw/day (male and female) and at 1000 mg/kg bw/day in male animals. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in these groups.
Exophthalmos – as described above - was observed in one control male animal (1/12) at the functional observations, too.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no mortality in 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was influenced by the test item in male and female animals at 1000 mg/kg bw/day.
The mean body weight was significantly reduced in male animals at 1000 mg/kg bw/day during the course of the entire study. The mean body weight gain was also significantly reduced in this group on Weeks 1, 2, 3, 4 and 7 and in the whole treatment period (between Day 0 and 50).
In females at 1000 mg/kg bw/day statistically significantly lower mean body weight gain was observed during the first week of the pre-mating period and during the gestation period between gestation days 7-14, 14-21 and in the total of 0 21. Similarly, the body weight was significantly lower than in the control group on gestation days 14 and 21. During the whole lactation period, the mean body weight was also statistically significantly lower in this group than in the control group.
There were no statistically significant differences between the control and test item treated groups in the mean body weight (males and females at 100 and 300 mg/kg bw/day). Some statistical significance was observed with respect to the control at the lower mean body weight gain of female animals at 300 mg/kg bw/day between gestation days 0-7 and 0-21.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was influenced by the test item in male and female animals at 1000 mg/kg bw/day.
The daily food mean consumption was statistically significantly reduced in male animals at 1000 mg/kg bw/day during the treatment period.
The mean food consumption was statistically significantly less than in the control group in female animals at 1000 mg/kg bw/day during the pre-mating period, in the first and second week of the gestation period and in the whole lactation period.
There were no test item related changes in the daily mean food consumption of male and female animals at 100 and 300 mg/kg bw/day.
At 300 mg/kg bw/day in male and female animals, reduced daily mean food consumption was noted when compared to the control on each measurement occasion, and in some cases attaining statistical significance: in males on Weeks 4 and 7, in females on the first and second gestation week. However these were minor differences with respect to their control and were judged toxicologically not relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day. Elevated mean platelet count in male animals at 300 and 1000 mg/kg bw/day was not accompanied by changes in parameters of hemostasis or histopathology lesions therefore its toxicological importance is equivocal.
Compared to their control group, the mean percentage of basophil granulocytes (BASO) were significantly lower in male animals at 100, 300 and 1000 mg/kg bw/day. The mean platelet counts (PLT) in males at 300 and 1000 mg/kg bw/day was statistically significantly higher compared to their control. In both groups all the individual values were out of the historical control range., hHowever, the detected alterations had no adverse effects, thus the changes were considered to be non-adverse.
In female animals, statistical significances were detected at the lower mean percentage of neutrophil granulocytes (NEU) and at the higher mean percentage of lymphocytes (LYM) at 300 mg/kg bw/day and at the lower mean percentage of monocytes (MONO) at 100 mg/kg bw/day when compared to their control. The red blood cell (erythrocyte) count (RBC) at 1000 mg/kg bw/day was slightly but statistically significantly higher than the in the control. The mean corpuscular (erythrocyte) volume (MCV), the mean Corpuscular (erythrocyte) hemoglobin (MCH) at 1000 mg/kg bw/day and the mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) at 300 mg/kg bw/day was slightly but statistically significantly lower than the in the control. The prothrombin time (PT) was statistically higher at 1000 mg/kg bw/day when compared to the control group. All these slight changes in female animals are considered to have no toxicological relevance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Evaluation of the clinical chemistry parameters did not reveal pathologic changes in the examined biochemical parameters. The mean activity of alanine aminotransferase and aspartate aminotransferase activity at 1000 mg/kg bw/day (male and female) slightly exceeded the control. However, in the lack of supporting histopathological lesions these were considered to be adaptive changes and not of toxicological relevance.
Statistically lower total bilirubin concentration (TBIL) at 100 and 1000 mg/kg bw/day, glucose concentration (GLUC) at 300 and 1000 mg/kg bw/day, albumin (ALB) and total protein (TPROT) concentrations at 1000 mg/kg bw/day were detected in males.

These changes were considered to have no toxicological relevance.
In the male animals at 1000 mg/kg bw/day, the mean alanine aminotransferase activity (ALT) and the aspartate aminotransferase activity (AST) was statistically significantly higher than in the control group (39 % and 23 %, respectively).
Similarly to males, in female animals, at 1000 mg/kg bw/day, the mean alanine aminotransferase activity (ALT) and the aspartate aminotransferase activity (AST) was statistically significantly higher than in the control group (33 % and 39 %, respectively).
Statistically lower albumin (ALB) and total protein (TPROT) concentrations at 100 and 1000 mg/kg bw/day were detected in females. These changes were considered to have no toxicological relevance.
Statistically significant difference with respect to the control was noted for the slightly lower thyroid hormone (free T4) level in male parental animal at 1000 mg/kg bw/day. This minor difference was considered to be toxicologically not relevant as most of individual values were within the historical control range (2.72±0.39 ng/dL; n=96; min: 1.91 ng/dL; max = 3.86 ng/dL). Moreover there were no histological changes in the organs of hypothalamic-pituitary-thyroid axis of examined animals at 1000 mg/kg bw/day
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of male or female animals at 1000 mg/kg/bw/day (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals; full histopathology in selected animals).

Histological examination revealed in the majority of control and treated male animals (8/12 control, 9/12 at 100 mg/kg bw/day, 11/12 at 300 mg/kg bw/day, 10/12 at 1000 mg/kg bw/day) minimal, mild or moderate degree vacuolation in the hepatocytes. This phenomenon could be considered as hepatic lipidosis.
Hepatic lipidosis could be considered as a light reversible liver injury and it could be in connection with a disturbance of energy metabolism of affected hepatocytes, probably dietetic origin.
The centrilobular vacuolation of hepatocytes was not observed in the liver of majority of female animals (2/12 control, 1/5 at 1000 mg/kg bw/day) and the occurrence was of minimal degree in all female cases.
In all male animals, the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well.
In the female animals the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. 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 was normal in all cases as well.
In some female animals (1/12 control; 1/1 at 100 mg/kg bw/day; 1/12 at 1000 mg/kg bw/day) dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
Alveolar emphysema in minimal or mild degree was observed in the lungs of male (1/5 control) and female (2/5 control) animals. Acute hemorrhage in the lung was noted in the lungs of male animals (1/5 control, 1/5 at 1000 mg/kg bw/day) This finding was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.

Hyperplasia of bronchus associated lymphoid tissue (BALT) was noted for male animals (3/5 control and 1/5 at 1000 mg/kg bw/day). Hyperplasia of BALT is a physiological immune-morphological phenomenon, without toxicological significance.
Acute hemorrhage in the thymus was observed in one male (1/5 control, 1/5 at 1000 mg/kg bw/day). This finding was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the animals.
The cytomorphology of the endocrine glands were the same in the control and the treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
TO BE ADDED
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Estrus cycle
An altered estrous cycle was detected at 1000 mg/kg bw/day.
During the pre-treatment period, there were no significant differences between the control and treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrous.
In the pre-mating period at 1000 mg/kg bw/day, statistically significant difference was observed when compared to their control in the lower number of animals with regular cycle. With this, a statistically significant lower number of cycles, lower mean number of days in pro-estrous, higher mean number of days in diestrous and correspondingly longer length of cycles was observed.

Delivery Data of Dams
Delivery data of dams was affected at 1000 mg/kg bw/day dose level. A higher percent and mean of pre-implantation loss and lower mean birth (total birth, live born and viable pups per litter) were observed at the high dose treated animals.
Statistically significant difference was observed at 1000 mg/kg bw/day when compared to their control in the lower number of implantations, mean number of total birth, liveborns and viable pups on day 0, in the higher number of post-implantation loss (total and mean) and in the longer pregnancy.
The number of pregnant females and dams delivered, the live birth index were comparable in all groups and the differences between control and test item treated doses did not obtain any statistical significance

Reproductive Performance
An influence on the reproductive performance was assumed at 1000 mg/kg bw/day as the mean number of pre-coital and conceiving days was higher than in the control group.
Statistically significant differences between the control and test item treated groups were detected in male animals at 100 mg/kg bw/day and in female animals at 100 and 1000 mg/kg bw/day at the higher fertility indices. One pair of control group mated without pregnancy resulting in lower fertility indices. Thus test item treated groups showed statistically significantly higher 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 and at the lower gestation index in female animals at 1000 mg/kg bw/day comparing to their control. In this group 1 out of 12 females were non-pregnant but mated (sperm positive). Considering the minor degree of these changes and the historical control data, these differences with respect to their control were not considered to be toxicologically relevant.
Statistically significantly higher mean conceiving days (5.0) were noted for the female animals in the 1000 mg/kg bw/day group. This value exceeded the mean historical control value (2.7 days, SD=1.4 days, n=144).

The mean number of pre-coital days was double of the actual control value and more than double of the historical control (1.7 days SD = 1.4 days; n= 162). Therefore, an influence due to test item treatment on the mean pre-coital interval and conceiving days can be assumed for the high dose level.
Statistically significance was observed at the lower mean number of conceiving days in female animals at 100 mg/kg bw/day. This finding was not considered to be toxicologically relevant.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: systemic toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Remarks on result:
other: fertility
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Table 5 Clinical signs noted during the course of the study

Observation

Pre-mating[bsc1] 

Post-mating

Gestation

Lactation

Salivation

0/12

2/2

3/10

1/10

Nuzzling up the bedding material

0/12

2/2

3/10

1/10

Piloerection

0/12

0/2

4/10

3/10

Decreased activity

0/12

0/2

1/10

1/10

Decreased body tone

0/12

0/2

0/10

1/10

Increased respiratory rate

0/12

0/2

0/10

1/10

Conclusions:
Under the conditions of the present study, the test item caused clinical signs (salivation, nuzzling up the bedding material, piloerection, decreased activity, decreased body tone or increased respiratory rate), reduced body weight and body weight gain, reduced food consumption in parental male and female Hsd.Han: Wistar rats at 1000 mg/kg bw/day administered by oral gavage.
As a consequence an influence on the estrous cycle, delivery data of dams (higher pre-implantation loss, lower birth mean), reproductive performance of male and female animals (prolonged pre-coital and conceiving periods) were also observed at 1000 mg/kg bw/day.
The development of the F1 offspring was also impaired at 1000 mg/kg bw/day due to poor general condition of the dams at this dose level: clinical signs with higher incidence, higher mortality and reduced body weight gain and body weight were observed with respect to their control.

Based on these observations the No Observed Adverse Effect Levels (NOAELs) were determined as follows:

NOAEL for systemic toxicity of male and female rats: 300 mg/kg bw/day
NOAEL for reproductive performance of male/female rats: 300 mg/kg bw/day
NOAEL for F1 Offspring: 300 mg/kg bw/day
Executive summary:

The purpose of this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was to obtain initial information on the toxic potential of the test item and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as development of the F1 offspring from conception to day 13 post-partum when repeatedly administered orally (by gavage) to parental animals at doses of 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day compared to control animals.

Four groups of Hsd.Han:Wistar rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, sunflower oil.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. The test item concentrations in the dosing formulations varied in the acceptable range between 96 % and 100 % of the nominal values confirming the proper dosing.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 51 days). Females were additionally exposed through the gestation period and up to lactation days 13-17, up to the day before necropsy (altogether for 51-71 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.

The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (T4) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) and pituitary in the control and high dose groups. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups. In addition, liver was also processed and examined histologically in all male animals due to necropsy findings observed in several male animals (pale, nutmeg-like pattern). Seminal vesicle (smaller than normal) of one male animal and uterus of one female (hydrometra) in the low dose group were also processed and examined histopathologically on the basis of necropsy finding.

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only. Historical control data were also considered.

               

There was no mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study. Test item related clinical signs were observed in male animals at 1000 mg/kg bw/day and in female animals at 300 and 1000 mg/kg bw/day at the daily clinical observations: salivation, nuzzling up the bedding material, piloerection, decreased activity in males and females at 1000 mg/kg bw/day and salivation, nuzzling up the bedding material, piloerection in females at 300 mg/kg bw/day. Piloerection or decreased activity was observed at 1000 mg/kg bw/day (male and female) and at 300 mg/kg bw/day also at the detailed weekly clinical observations. Additionally, decreased body tone and increased respiratory rate was noted for one female animal at 1000 mg/kg bw/day on the day of the delivery. Functional observations revealed piloerection in one female animal at 1000 mg/kg bw/day group. The body weight gain and body weight werely reduced in male and female animals at 1000 mg/kg bw/day. The food consumption was lower than in the control group in male and female animals at 1000 mg/kg bw/day.

An influence on the estrous cycle was detected at 1000 mg/kg bw/day. Lower number of animals with regular cycle, lower number of cycles and lower mean number of days in pro-estrous, higher mean number of days in diestrous and longer length of cycles were observed. An influence on the reproductive performance was assumed at 1000 mg/kg bw/day as the mean number of pre-coital and conceiving days was higher than in the control group. This finding is in line with the altered estrous cycle. Higher percent and mean of preimplantation loss and lower mean birth (total birth, live born and viable pups per litter) were observed at the high dose treated animals. This may be associated with of the reduced food consumption and consequently decreased body weight gain followed by decreased absolute body weight observed at the high dose level. There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day. Elevated mean platelet count in male animals at 300 and 1000 mg/kg bw/day was not accompanied by changes in parameters of hemostasis or histopathology lesions. Therefore, its toxicological importance is equivocal.

Clinical chemistry investigations did not reveal toxic changes examined parameters at 100, 300 or 1000 mg/kg bw/day. The mean activity of alanine aminotransferase and aspartate aminotransferase activity at 1000 mg/kg bw/day (male and female) slightly exceeded the control. However, in the lack of supporting histopathological lesions these were considered to be adaptive changes. The slightly lower mean thyroid hormone (free T4) level in parental male animal at 1000 mg/kg bw/day was considered to be not relevant as there were no histological changes in the organs of hypothalamic-pituitary-thyroid axis of examined animals and most of the individual values still remained within the historical control range.

Specific macroscopic alterations related to the test item were not found at necropsy.

Pale and nutmeg-like patterned liver noted for several male animals at 100, 300 and 1000 mg/kg bw/day were judged to be not related to the test item because the incidence of these hepatic changes was not related to doses, moreover histopathological examinations revealed vacuolation in the hepatocytes, (indicative of hepatic lipidosis) with similar incidence in control and test item treated 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.

The examined organ weights of animals selected for toxicity examinations were comparable in the control and 100, 300 or 1000 mg/kg bw/day test item treated groups at the end of the treatment period. Histopathological examinations of the investigated organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day. There were no changes related to the test item in the organs or tissues of selected animals (male or female) at 1000 mg/kg bw/day.

          

The percentage of offspring with signs (cold, not suckled, smaller than normal) was higher than in the control group at 1000 mg/kg bw/day. The number of dead pups was also higher in this group than in the control and in the lower doses. The absolute anogenital distances were slightly shorter with respect to their control at 1000 mg/kg bw/day (male and female). However, when corrected for respective body weight there was no biologically significant difference to the control. The offspring’s body weight development was depressed at 1000 mg/kg bw/day between postnatal days 0 and 13.

Under the conditions of the present study, the test item caused clinical signs (salivation, nuzzling up the bedding material, piloerection, decreased activity, decreased body tone or increased respiratory rate), reduced body weight and body weight gain, reduced food consumption in parental male and female Hsd.Han: Wistar rats at 1000 mg/kg bw/day administered by oral gavage. A further influence on the estrous cycle, delivery data of dams (higher pre-implantation loss, lower birth mean), reproductive performance of male and female animals (prolonged pre-coital and conceiving periods) were also observed at 1000 mg/kg bw/day. No correlating histopathological findings were obtained in the respective reproductive organs. The development of the F1 offspring was also impaired at 1000 mg/kg bw/day: clinical signs with higher incidence, reduced body weight gain and body weight. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

 

NOAEL for systemic toxicity of male and female rats:               300 mg/kg bw/day

NOAEL for reproductive performance of male/female rats:     300 mg/kg bw/day

NOAEL for F1 Offspring:      300 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline compliant study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline study.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

OECD 422

The purpose of this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was to obtain initial information on the toxic potential of the test item and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as development of the F1 offspring from conception to day 13 post-partum when repeatedly administered orally (by gavage) to parental animals at doses of 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day compared to control animals.

Four groups of Hsd.Han:Wistar rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, sunflower oil.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. The test item concentrations in the dosing formulations varied in the acceptable range between 96 % and 100 % of the nominal values confirming the proper dosing.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 51 days). Females were additionally exposed through the gestation period and up to lactation days 13-17, up to the day before necropsy (altogether for 51-71 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.

The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (T4) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) and pituitary in the control and high dose groups. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups. In addition, liver was also processed and examined histologically in all male animals due to necropsy findings observed in several male animals (pale, nutmeg-like pattern). Seminal vesicle (smaller than normal) of one male animal and uterus of one female (hydrometra) in the low dose group were also processed and examined histopathologically on the basis of necropsy finding.

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only. Historical control data were also considered.

               

There was no mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study. Test item related clinical signs were observed in male animals at 1000 mg/kg bw/day and in female animals at 300 and 1000 mg/kg bw/day at the daily clinical observations: salivation, nuzzling up the bedding material, piloerection, decreased activity in males and females at 1000 mg/kg bw/day and salivation, nuzzling up the bedding material, piloerection in females at 300 mg/kg bw/day. Piloerection or decreased activity was observed at 1000 mg/kg bw/day (male and female) and at 300 mg/kg bw/day also at the detailed weekly clinical observations. Additionally, decreased body tone and increased respiratory rate was noted for one female animal at 1000 mg/kg bw/day on the day of the delivery. Functional observations revealed piloerection in one female animal at 1000 mg/kg bw/day group. The body weight gain and body weight werely reduced in male and female animals at 1000 mg/kg bw/day. The food consumption was lower than in the control group in male and female animals at 1000 mg/kg bw/day.

An influence on the estrous cycle was detected at 1000 mg/kg bw/day. Lower number of animals with regular cycle, lower number of cycles and lower mean number of days in pro-estrous, higher mean number of days in diestrous and longer length of cycles were observed. An influence on the reproductive performance was assumed at 1000 mg/kg bw/day as the mean number of pre-coital and conceiving days was higher than in the control group. This finding is in line with the altered estrous cycle. Higher percent and mean of preimplantation loss and lower mean birth (total birth, live born and viable pups per litter) were observed at the high dose treated animals. This may be associated with of the reduced food consumption and consequently decreased body weight gain followed by decreased absolute body weight observed at the high dose level. There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day. Elevated mean platelet count in male animals at 300 and 1000 mg/kg bw/day was not accompanied by changes in parameters of hemostasis or histopathology lesions. Therefore, its toxicological importance is equivocal.

Clinical chemistry investigations did not reveal toxic changes examined parameters at 100, 300 or 1000 mg/kg bw/day. The mean activity of alanine aminotransferase and aspartate aminotransferase activity at 1000 mg/kg bw/day (male and female) slightly exceeded the control. However, in the lack of supporting histopathological lesions these were considered to be adaptive changes. The slightly lower mean thyroid hormone (free T4) level in parental male animal at 1000 mg/kg bw/day was considered to be not relevant as there were no histological changes in the organs of hypothalamic-pituitary-thyroid axis of examined animals and most of the individual values still remained within the historical control range.

Specific macroscopic alterations related to the test item were not found at necropsy.

Pale and nutmeg-like patterned liver noted for several male animals at 100, 300 and 1000 mg/kg bw/day were judged to be not related to the test item because the incidence of these hepatic changes was not related to doses, moreover histopathological examinations revealed vacuolation in the hepatocytes, (indicative of hepatic lipidosis) with similar incidence in control and test item treated 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.

The examined organ weights of animals selected for toxicity examinations were comparable in the control and 100, 300 or 1000 mg/kg bw/day test item treated groups at the end of the treatment period. Histopathological examinations of the investigated organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day. There were no changes related to the test item in the organs or tissues of selected animals (male or female) at 1000 mg/kg bw/day.

          

The percentage of offspring with signs (cold, not suckled, smaller than normal) was higher than in the control group at 1000 mg/kg bw/day. The number of dead pups was also higher in this group than in the control and in the lower doses. The absolute anogenital distances were slightly shorter with respect to their control at 1000 mg/kg bw/day (male and female). However, when corrected for respective body weight there was no biologically significant difference to the control. The offspring’s body weight development was depressed at 1000 mg/kg bw/day between postnatal days 0 and 13.

Under the conditions of the present study, the test item caused clinical signs (salivation, nuzzling up the bedding material, piloerection, decreased activity, decreased body tone or increased respiratory rate), reduced body weight and body weight gain, reduced food consumption in parental male and female Hsd.Han: Wistar rats at 1000 mg/kg bw/day administered by oral gavage. A further influence on the estrous cycle, delivery data of dams (higher pre-implantation loss, lower birth mean), reproductive performance of male and female animals (prolonged pre-coital and conceiving periods) were also observed at 1000 mg/kg bw/day. No correlating histopathological findings were obtained in the respective reproductive organs. The development of the F1 offspring was also impaired at 1000 mg/kg bw/day: clinical signs with higher incidence, reduced body weight gain and body weight. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

 

NOAEL for systemic toxicity of male and female rats: 300 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 300 mg/kg bw/day

NOAEL for F1 Offspring: 300 mg/kg bw/day

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item is not classified for toxicity to reproduction or developmental toxicity / teratogenicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.