Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Based on the results of an OECD 422 compliant study, a NOAEL for systemic as well as for fertility effects was determined to be 1000 mg/kg bw/d since no adverse effects were observed.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-09-17 to 2020-02-24
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
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: in house bred
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) x 12 - 13 wks
- Weight at study initiation: (P) Males: 343 - 396 g; Females: 209 - 239 g
- Fasting period before study: no
- Housing:
Before mating: 2 animals of the same sex/cage
Mating hours: 1 male and 1 female/cage
Pregnant females were housed individually
Males after mating: 2 animals/cage

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days

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:

VEHICLE
- Justification for use and choice of vehicle: the test item is not stable in water and sunflower oil is well tolerated and accepted by the guideline
- Concentration in vehicle: 0, 20, 60, 200 mg/mL
- Amount of vehicle: 5 mL/kg bw/d
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- 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:
The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity).
The concentration of the test item in the dosing formulations used for animal’s treatment was checked two times during the study. Concentration of the test item in the dosing formulations varied between the range of 92.5 % and 101 % of the nominal values and formulations were homogenous thereby confirming the proper dosing.
Duration of treatment / exposure:
males: 42 days
females: up to 51-62 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
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
yes, historical
Details on study design:
- Dose selection rationale: based on preliminary dose range finder and up to the limit dose recommended by the guideline (i. e. 1000 mg/kg bw/d)
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: times of weekly weighing
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 (Day 0) and then weekly, gestation days 0, 7, 14, 21 and post-partum day 0, 4, 13.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: by visual inspection
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment started 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 was also prepared on the day of the necropsy. Vaginal smears were stained with 1 % aqueous methylene blue solution and examined with a light microscope after drying.
Sperm parameters (parental animals):
Parameters examined in all male parental animals:
testis weight, epididymis weight, sperm count in testes
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), pup weight on the day of 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 as soon as possible after the last litters in each generation were produced
- Maternal animals: All surviving animals after the last litter of each generation was weaned

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in below were prepared for microscopic examination and weighed, respectively:
Adrenal glands
Aorta
Bone with marrow and joint (femur)
Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
Eyes (lachrymal gland with Harderian glands)
Gonads (testes with epididymides, ovaries, uterus with vagina)
Heart
Kidneys
Large intestines (caecum, colon, rectum),
Liver
Lungs (with main stem bronchi; inflation with fixative and then immersion;)
Lymph nodes (submandibular, mesenteric)
Mammary gland
Muscle (quadriceps)
Esophagus
Pancreas
Pituitary
Prostate
Salivary glands (submandibular)
Sciatic nerve
Seminal vesicle with coagulating gland
Skin
Small intestines (duodenum, ileum, jejunum; including Peyer’s patches)
Spinal cord (at three levels: cervical, mid-thoracic and lumbar)
Spleen
Sternum
Stomach
Thymus
Thyroid + parathyroid
Trachea
Urinary bladder
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 14 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
Statistics:
The statistical evaluation of appropriate data (marked with † above) 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.
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:
no effects observed
Description (incidence and severity):
Adverse signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (100, 300 and 1000 mg/kg bw/day, male or female).
Darker than normal and wet bedding material, nuzzling up the bedding material and salivation were observed at 1000 mg/kg bw/day as treatment related findings, which however were considered to be not adverse.
Darker than normal and wet bedding material was presumably indicative of diuresis. However, there were no related findings in clinical chemistry parameters or in kidney organ pathology. Nuzzling up the bedding material and salivation were detected shortly after the administration and ceased within a short time period and .is likely caused by the irritant character of the test item. Therefore, these observations were considered to be toxicologically not relevant.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality in 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was not adversely affected by the test item in male or female animals administered with 100, 300 or 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not adversely affected in male or female animals at 100, 300 and 1000 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
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 or blood coagulation parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The examined clinical chemistry parameters were not adversely affected in male and female animals at 100, 300 or 1000 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Detailed weekly and Functional observations:
The behavior and physical condition of animals was not adversely affected by the test item at any dose level (100, 300 or 1000 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.
All animals were normal in control, 100, 300 and 1000 mg/kg bw/day groups (male and female) at the detailed weekly clinical observations.
Functional observations did not demonstrate any test item related adverse changes. Signs of diuresis (dark and wet bedding material) was detected in cages of all animals at 1000 mg/kg bw/day).
The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (control, 100, 300 and 1000 mg/kg bw/day; n=5 animals/sex/group).


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 investigations did not reveal any toxic or other test item related lesions detectable by microscopic examination of the investigated reproductive and other organs of male and female animals at 1000 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The examined parameters of the estrous cycle were comparable in the control and test item administered animals (100, 300 and 1000 mg/kg bw/day).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, prostate, seminal vesicles, and coagulating glands was normal in all cases as well (12/12 control, 1/1 at 300 mg/kg bw/day, 12/12 at 1000 mg/kg bw/day).
Reproductive performance:
no effects observed
Description (incidence and severity):
The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 100, 300 or 1000 mg/kg bw/day.
Please refer to result tables attached.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item related clinical signs in the offspring from post-natal day 0 to 13.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
The extrauterine mortality was low and comparable in the control, 100, 300 and 1000 mg/kg bw/day from birth to post-natal day 13.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development of the offspring was undisturbed at 100, 300 and 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances were not affected by the test item at 100, 300 and 1000 mg/kg bw/day in male or female offspring.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on post-natal day 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Please refer to attached result tables.

Conclusions:
Under the conditions of this OECD 422 compliant study, the test item at 100, 300 or 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats as investigated in this study. There were no signs of systemic toxicity in selected male or female animals at 100, 300 or 1000 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/ female rats: 1000 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 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 according to OECD 422.

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

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity).

The concentration of the test item in the dosing formulations used for animal’s treatment was checked two times during the study. Concentration of the test item in the dosing formulations varied between the range of 92.5 % and 101 % of the nominal values and formulations were homogenous thereby 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 42 days). Dams were additionally exposed through the gestation period and up to lactation days 13-15 (altogether for 51-61 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 and on the day of the necropsy.

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

Blood samples were collected for possible determination of serum levels of thyroid hormones (FT3, FT4, TSH) from 2-8 pups per litter (where it was feasible) on post-natal day 4, from all dams and from 4-7 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. Adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed for five male and five female animals randomly selected from each group.

Histopathology examination was performed on reproductive organs (testes, epididymides, prostate, and seminal vesicles with coagulating glands, uterus with oviduct, cervix, vagina and ovaries) in the control and high dose groups and in not delivered pregnant female animals and the male this female cohabited with in the mid dose group.

Full histopathology examinations were 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, organs showing macroscopic changes (kidneys, spleen) were also processed and examined histologically in animals in low or mid dose groups. 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 test item related mortality in 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of study.

Adverse signs of systemic toxicity related to the test item were not detected at any dose level (100, 300 or 1000 mg/kg bw/day, male or female) at the daily or detailed weekly clinical observations or at the terminal functional observations.

Test item related clinical signs – darker than normal and wet bedding material, nuzzling up the bedding material (male and female) and salivation (male) – were observed at 1000 mg/kg bw/day. Darker than normal and wet bedding material – indicative of diuresis – was detected in each cage of animals administered with 1000 mg/kg bw/day from Day 5 up to the end of the treatment period (pre-mating, mating and post-mating period in male animals, pre-mating, mating, gestation and lactation periods in dams). In the lack of related findings in clinical chemistry parameters or in organ pathology, these findings were considered to be not adverse ones. Salivation and nuzzling up the bedding material were seen transiently and were with short duration.

The mean body weight gain and mean body weight were not influenced by the test item in male or female animals administered with 100, 300 or 1000 mg/kg bw/day. Minor changes in the body weight gain detected in the male animals at 1000 mg/kg bw/day during the pre-mating period and if calculated for the whole study did not result in significant changes in the body weight of these animals, therefore were considered to be toxicologically not relevant.

The food consumption was not adversely affected in male or female animals at 100, 300 or 1000 mg/kg bw/day. Slightly reduced mean daily food consumption in male animals at 1000 mg/kg bw/day during the pre-mating period in accordance with the body weight gain changes were judged to be toxicologically not relevant. A test item influence on the estrous cycle was not detected at any dose level (100, 300 or 1000 mg/kg bw/day).

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

Delivery data of dams was not adversely affected at 100, 300 or 1000 mg/kg bw/day 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.

The examined clinical chemistry parameters were not affected in male and female animals at 100, 300 or 1000 mg/kg bw/day.

The FT3 and FT4 thyroid hormone levels were not adversely affected in the parental male animals or in PN13 offspring at any dose levels. The TSH level was slightly elevated in PN13 offspring at all dose levels, however, without reaching statistical significance and no clear dose-response relationship.

Macroscopic alterations related to the effect of the test item were not detected in male or female animals at 100, 300 or 1000 mg/kg bw/day at the terminal necropsy.

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

The weights of examined organs (absolute, relative to body and brain weights) were not adversely affected by the test item in selected animals at 100, 300 or 1000 mg/kg bw/day.

Slight changes in the kidney weights relative to body weight in male animals at 1000 mg/kg bw/day were presumably related to the diuresis i.e. enhanced function of the organ. There were no related changes in clinical chemistry parameters or histopathology investigations. Therefore, this change in the kidney weight was considered to have little or no toxicological relevance.

Thymus weight (absolute and relative to body and brain weight) were statistically significantly decreased in male animals of the high and low dose group, respectively. A decreased thymus weight was also recorded in the mid dose males, however, not reaching stat. significance. Changes in thymus weight could be associated with increased white blood cell count of male animals being indicative for increased immunesystem activity. However, in the absence of histopathological findings this observation was not regarded adverse.

Histopathological examinations of the investigated sexual organs and accessory sex 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 or in not delivered female animal or in its male partner at 300 mg/kg bw/day.

Histopathological investigations did not reveal any toxic or other test item related lesions detectable by microscopic examination of the investigated organs of selected male and female animals at 1000 mg/kg bw/day.

The offspring’s development was not adversely influenced at any dose level. No effects on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

 

In conclusion, under the conditions of the present study, the test item did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 100, 300 or 1000 mg/kg bw/day by oral gavage as investigated in this study. There were no signs of systemic toxicity in selected male or female animals at 100, 300 or 1000 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.

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

NOAEL for systemic toxicity of male/female rats:             1000 mg/kg bw/day

NOAEL for reproductive performance of male/female rats:        1000 mg/kg bw/day

NOAEL for F1 Offspring:               1000 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and Guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
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 according to OECD 422.

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

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity).

The concentration of the test item in the dosing formulations used for animal’s treatment was checked two times during the study. Concentration of the test item in the dosing formulations varied between the range of 92.5 % and 101 % of the nominal values and formulations were homogenous thereby 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 42 days). Dams were additionally exposed through the gestation period and up to lactation days 13-15 (altogether for 51-61 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 and on the day of the necropsy.

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

Blood samples were collected for possible determination of serum levels of thyroid hormones (FT3, FT4, TSH) from 2-8 pups per litter (where it was feasible) on post-natal day 4, from all dams and from 4-7 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. Adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed for five male and five female animals randomly selected from each group.

Histopathology examination was performed on reproductive organs (testes, epididymides, prostate, and seminal vesicles with coagulating glands, uterus with oviduct, cervix, vagina and ovaries) in the control and high dose groups and in not delivered pregnant female animals and the male this female cohabited with in the mid dose group.

Full histopathology examinations were 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, organs showing macroscopic changes (kidneys, spleen) were also processed and examined histologically in animals in low or mid dose groups. 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 test item related mortality in 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of study.

Adverse signs of systemic toxicity related to the test item were not detected at any dose level (100, 300 or 1000 mg/kg bw/day, male or female) at the daily or detailed weekly clinical observations or at the terminal functional observations.

Test item related clinical signs – darker than normal and wet bedding material, nuzzling up the bedding material (male and female) and salivation (male) – were observed at 1000 mg/kg bw/day. Darker than normal and wet bedding material – indicative of diuresis – was detected in each cage of animals administered with 1000 mg/kg bw/day from Day 5 up to the end of the treatment period (pre-mating, mating and post-mating period in male animals, pre-mating, mating, gestation and lactation periods in dams). In the lack of related findings in clinical chemistry parameters or in organ pathology, these findings were considered to be not adverse ones. Salivation and nuzzling up the bedding material were seen transiently and were with short duration.

The mean body weight gain and mean body weight were not influenced by the test item in male or female animals administered with 100, 300 or 1000 mg/kg bw/day. Minor changes in the body weight gain detected in the male animals at 1000 mg/kg bw/day during the pre-mating period and if calculated for the whole study did not result in significant changes in the body weight of these animals, therefore were considered to be toxicologically not relevant.

The food consumption was not adversely affected in male or female animals at 100, 300 or 1000 mg/kg bw/day. Slightly reduced mean daily food consumption in male animals at 1000 mg/kg bw/day during the pre-mating period in accordance with the body weight gain changes were judged to be toxicologically not relevant. A test item influence on the estrous cycle was not detected at any dose level (100, 300 or 1000 mg/kg bw/day).

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

Delivery data of dams was not adversely affected at 100, 300 or 1000 mg/kg bw/day 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.

The examined clinical chemistry parameters were not affected in male and female animals at 100, 300 or 1000 mg/kg bw/day.

The FT3 and FT4 thyroid hormone levels were not adversely affected in the parental male animals or in PN13 offspring at any dose levels. The TSH level was slightly elevated in PN13 offspring at all dose levels, however, without reaching statistical significance and no clear dose-response relationship.

Macroscopic alterations related to the effect of the test item were not detected in male or female animals at 100, 300 or 1000 mg/kg bw/day at the terminal necropsy.

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

The weights of examined organs (absolute, relative to body and brain weights) were not adversely affected by the test item in selected animals at 100, 300 or 1000 mg/kg bw/day.

Slight changes in the kidney weights relative to body weight in male animals at 1000 mg/kg bw/day were presumably related to the diuresis i.e. enhanced function of the organ. There were no related changes in clinical chemistry parameters or histopathology investigations. Therefore, this change in the kidney weight was considered to have little or no toxicological relevance.

Thymus weight (absolute and relative to body and brain weight) were statistically significantly decreased in male animals of the high and low dose group, respectively. A decreased thymus weight was also recorded in the mid dose males, however, not reaching stat. significance. Changes in thymus weight could be associated with increased white blood cell count of male animals being indicative for increased immunesystem activity. However, in the absence of histopathological findings this observation was not regarded adverse.

Histopathological examinations of the investigated sexual organs and accessory sex 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 or in not delivered female animal or in its male partner at 300 mg/kg bw/day.

Histopathological investigations did not reveal any toxic or other test item related lesions detectable by microscopic examination of the investigated organs of selected male and female animals at 1000 mg/kg bw/day.

The offspring’s development was not adversely influenced at any dose level. No effects on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

 

In conclusion, under the conditions of the present study, the test item did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 100, 300 or 1000 mg/kg bw/day by oral gavage as investigated in this study. There were no signs of systemic toxicity in selected male or female animals at 100, 300 or 1000 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.

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

NOAEL for systemic toxicity of male/female rats:             1000 mg/kg bw/day

NOAEL for reproductive performance of male/female rats:        1000 mg/kg bw/day

NOAEL for F1 Offspring:               1000 mg/kg bw/day

Effects on developmental toxicity

Description of key information

Based on the results of an OECD 422 compliant study, a NOAEL for developmental toxicity was determined to be 1000 mg/kg bw/d since no adverse effects were observed.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

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 on repeated dose toxicity the test item does not require classification as toxic for reproduction according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.