Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(9,9-Dibutyl-9H-fluoren-2-yl)-2-methyl-2-morpholin-4-yl-propan-1-one
Cas Number:
2020359-04-8
Molecular formula:
C29H39NO2
IUPAC Name:
1-(9,9-Dibutyl-9H-fluoren-2-yl)-2-methyl-2-morpholin-4-yl-propan-1-one
Test material form:
solid: particulate/powder
Details on test material:
a off-white powdery solid, without irritating odor

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals were housed in TECNIPLAST cages 1500U from the Tecniplast Company, Italy. The cages have high density polypropylene body, measuring 480 x 375 x 210 mm - floor area 1500 cm2. The cages, cage racks and other equipment were sanitized according to standard operation procedures.
On arrival, animals were placed into the cages, 5 rats per cage.
48-hours after arrival animals were weighted and kept in their cages until the start of the study to allow for acclimation to the animal room conditions, which are identical as defined for the experimental part of the study. After randomisation were placed three and two rats of the same sex.
The study was carried out in the experimental animal house of the Test Site with the pressure air-condition system. The animals were housed in one room under the defined laboratory conditions. The temperature and relative humidity in the animal room were recorded daily (dataloger No.: TH 464); mean values of temperature were 22.35±0.83°C at mean relative humidity 48.16±6.04% (mean±SD).
The artificial lighting was setting in a 12-hour light and 12-hour dark photoperiod.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Duration of treatment / exposure:
The animals were dosed daily for 7 days a week.
Frequency of treatment:
Once per day.
Females were treated during:
• 14-day pre-mating,
• 14-day mating (maximum)
• 22-day gestation (approximately)
• 13-day lactation
Females with no evidence of copulation and that failed to deliver were dosed for a total of 53 doses.
Males were treated during:
• 14-day pre-mating,
• 14-day mating (maximum)
Doses / concentrationsopen allclose all
Dose / conc.:
75 mg/kg bw/day
Remarks:
Low
Dose / conc.:
250 mg/kg bw/day
Remarks:
Mid
Dose / conc.:
750 mg/kg bw/day
Remarks:
High
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
General clinical observations were performed once a day 2 hours after dosing. Detailed clinical observations were made in all animals prior to the first exposure, and at a week thereafter. The signs as changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity as well as changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies were recorded.
All animals were checked for morbidity or mortality at least twice daily. The health condition of the animals, behaviour, reaction of the animals to the applied test item, their well-being were monitored and recorded in raw data.

Rats were inspected daily for test item-related morbidity and mortality.

Body weight of adult animals was evaluated on the first day of dosing, weekly thereafter, and at termination. During pregnancy, females were weighed on Days 0, 7, 14 and 20 and within 24 hours of parturition (Day1 post-partum), and Day 4 and Day 13 post-partum. These observations were reported individually for each adult animal.
Live pups weight was recorded within 24 hours of parturition, Day 4 and Day 13 post-partum.

Food consumption were measured weekly.
Sperm parameters (parental animals):
Detailed histological examination was performed on the testes, epididymides, seminal vesicles, prostate and thyroid glands of males of the highest dose group and the control group and on ovaries, uterus, cervix, vagina and thyroid gland of females of the highest dose group and the control group. The listed tissues were trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.
Postmortem examinations (parental animals):
At the end of the study, the animals were subjected to gross pathology examination. Special attention was paid to the reproductive organs. All specified organs were examined macroscopically and together with organs showing macroscopic lesions were collected, preserved in fixative solution (10 % buffered formalin / modified Davidson fixative) and stored for histological processing and analysis. Before euthanasia of adult animals, the blood samples were taken from vena caudalis for plasma thyroid hormone analysis (EDTA plasma; T4).
Males:
Specifically, in adult males after 28-day treatment, following organs were weighted at scheduled necropsy: testes, epididymides, prostate, seminal vesicles and coagulating glands, levator ani plus bulbocavernosus muscles (LABC complex), Cowper`s glands, glans penis, brain and thyroid with parathyroids (last one after fixation to avoid tissue damage). The testes, epididymides, prostate, seminal vesicles and coagulating glands, thyroid with parathyroid were collected and placed in the fixative.
Females:
The females that delivered were euthanized on Day 13 of lactation together with their litters. Vaginal smears were taken in the morning on day of necropsy to determine the stage of oestrous cycle and to correlate findings with histopathology of female reproductive organs. The ovaries with oviducts, uterus including cervix, brain and thyroid with parathyroids (after fixation) were weighted. The ovaries with oviducts, vagina and uterus including cervix were collected and fixed. At time of necropsy, the number of implantation sites in the uterus and the number of corpora lutea (CL) (CL of gestation and CL of lactation) in ovaries were counted and recorded for purpose of quantifying pre- and post-implantation loss. The uteri of females which have not delivered and appeared macroscopically non-gravid were opened and placed in 2% sodium hydroxide solution for detection of early implantation loss.
Postmortem examinations (offspring):
The pups found dead on Day 0-1 post-partum were examined macroscopically and their lungs were removed and placed in a saline-filled jar. The lungs being sinking to the bottom indicated delivery of stillborn pup. If the lungs were floating and/or there were the presence of milk in stomach, the pups were recorded as born alive.
On Day 13 after birth, one male and one female from each litter were used for thyroid evaluation. The blood of these pups was collected after decapitation and processed and stored at -20°C until thyroid hormone analysis. The pups were subjected to a gross necropsy with particular attention to developing external reproductive genitals. The thyroid glands were preserved and weighted after fixation. All other surviving pups were examined for gross abnormalities. Number of nipples / areolae was counted and recorded in male pups.
Statistics:
Individual quantitative data were summarized and analysed using descriptive statistics. This was done separately for males and females. The differences between the component groups were assessed statistically using appropriate parametric and/or non-parametric methods. The categorical data were evaluated applying contingency tables.
Significance level of 0.05 was considered to make relevant statistical conclusions.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No changes in gait, posture and response to handling as well as the presence of clonic or tonic movements or bizarre behaviour (self-mutilation, walking backwards) were observed
Mortality:
no mortality observed
Description (incidence):
No moribund animals were observed, and no test item-related mortality was recorded during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight of adult male as well as female rats increased in time in all experimental groups. Kruskal-Wallis test used for the analysis did not reject null hypothesis. There were no statistically significant differences amongst the treated groups at the significant level α = 0.05.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not observed during period of mating.
In summary, decreased food consumption in significant extent in high dose group of female rats were not accompanied with changes in the body weights and was not considered to be of toxicological relevance.
Organ weight findings including organ / body weight ratios:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The examination of oestrous cycle stage at necropsy day, Day 13 post-partum, showed the pattern characterizing the diestrous stage typical for suckling mothers.
No statistical differences when compared to the corresponding control animals were recorded in number of corpora lutea, in number of implantation sites per dam, in number of live pups per dam at birth as well as on Day 4 post-partum, in non-standardized litter weight at birth and on Day 4 after birth, in sex ratio (males/females) at Day 4 post-partum, in number of abnormal pups as well as in the loss of offspring.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Observed significant changes of AGI in female pups exposed prenatally to mid dose of test item without a corresponding change in growth (statistically highest mean body weight at Day 0) might reflect specific developmental effects. Altered hormone status accompanied with masculinization of female rat offspring may be excluded due to the opposite nature of the change - significant reduction of AGD/AGI.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
In case of the offspring, there were found one dead male pup in the control group (one pup for each mother; mothers No. 54 and 62, respectively) and two pups (one male and one female) in high dose group (mother No. 82). These pups were evaluated as dead pups (but alive at birth) based on the presence of milk in the stomach and floating lung in the saline solution. In the high dose group was also observed one stillborn female pup (mother No. 92) without milk gastric content and with lung falling in the saline solution. During pup counting on Day 4 after birth (before culling) one female pup was missing in the mid-dose group (mother No. 69).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significantly increased birth weights of male and female pups in the mid-dose group cannot be attributed to account of the extended intrauterine development, because no significant differences in gestation length were observed.
Under defined test conditions, the test item, specifically in the mid-dose group, induced alteration of physical development presented consequently as decrease of pup body weight in the lactation period dose- and gender dependently.
Statistically significant changes in offspring body weights might be considered as adverse in the sense of non-specified modulation effect of test item.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Observed significant changes of AGI in female pups exposed prenatally to mid dose of test item without a corresponding change in growth (statistically highest mean body weight at Day 0) might reflect specific developmental effects. Altered hormone status accompanied with masculinization of female rat offspring may be excluded due to the opposite nature of the change - significant reduction of AGD/AGI.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 750 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: reproduction toxicity

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
The test item 1-(9,9-Dibutyl-9H-fluoren-2-yl)-2-methyl-2-morpholin-4-yl-propan-1-one
- did not cause mortality of animals
- had no visible toxic effect on animals
- did not cause test item related alterations of organ and tissues. Macroscopic findings observed during gross necropsy (being sporadic incidence and without test item dose dependence) after histopathological evaluations were considered not to be test item related in the sense of the prevalence, severity and character.
- did not cause histopathological changes on examined reproduction organs
Statistically significant changes of relative organ weights observed in the testes, epididymis (right), thyroid glands and uteri including cervix based on the histological examinations revealed no serious morphological alterations and statistically significant differences were considered to be findings without biological/toxicological significance.
- did not show any effect on the reproductive performance of the exposed animals nor endocrine disrupting potential.
- caused dose-dependent decrease (both in offspring males and females) at 0.05 significance level of the body weight variable measured at Day 13 post-partum
- caused statistically significant decrease of body weight (both in offspring males and females) at the high dose (750 mg/kg) at Day 13 post-partum.
The statistically significant gender- and dose specific changes observed in the body weights of offspring might be considered as adverse in the sense of non-specified modulation effect of test item.
No-observed-adverse-effect level (NOAEL)
Based on the reproduction toxicity
• NOAEL for females and males was concluded to be 750 mg/kg