Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
a off-white powdery solid, without irritating odor

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar rats (76 total; 38/sex) were randomly assigned to one of four groups (5-7 sex/group) and administered with Olive oil (control) or 1-(9,9-Dibutyl-9H-fluoren-2-yl)-2-methyl-2-morpholin-4-yl-propan-1-one at dose levels of 100, 500 and 1000 mg/kg/day for 28 consecutive days by oral gavage.
The administered volume was 5 mL/kg body weight of the rat.
Sex:
male/female
Details on test animals 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.
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 with the pressure air-condition system. The animals were housed in two rooms under the same conditions. No other test item was applied in these rooms. The temperature and relative humidity in the animal room were recorded daily; mean values of temperature were 20-23.9°C and 41.5-50.6% relative humidity. In few cases the temperature did exceed the 24°C. If the temperature did exceed the 24°C, it was only rare and short-lasting deviations without impact on course of the study. The animals were subjected to a 12-hour light - 12-hour dark cycle.
The sanitation was performed according to standard operation procedures.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
Dose volume was 5.0 ml/kg of body weight and was adjusted according to the weight development of the animals.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Once per day, 28 days. The animals designated for post-treatment observation (5 animals per sex in control and high groups, respectively) remained untreated for subsequent 14 days.
Frequency of treatment:
daily
Doses / concentrations
Dose / conc.:
5 mg/kg bw (total dose)
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Blood samples were collected from fasted animals; haematology parameters were analysed at the same day as blood was collected from tail vein, clinical chemistry parameters were collected from vena cava caudalis, biological samples (plasma/serum) were stored according relevant condition for later analysis.
Plasma sample were retained for T4 determination if there is an indication for an effect on the pituitary-thyroid axis. The samples were stored at -20°C. Plasma samples for evaluation of APTT (Activated Partial Thromboplastin Time) were frozen at -20°C until to the analysis.
The blood collection, processing of the blood and the determination of its haematological and clinical chemistry parameters were performed according to the standard operation procedures.
The blood collection in all animals was done at prior to euthanasia of the animals.
The urinalysis was performed on urine collected from fasted animals; in deep anaesthesia immediately before the necropsy. Urine was collected directly from urinary bladder by needle and syringe.
Samples were frozen at -20°C until to the analysis. Following parameters were evaluated: specific gravity leucocytes, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, colour, clarity.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Rats were inspected twice daily for sings of toxicity. No deviations from normal findings in terms of: clinical signs, sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli), changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity as lacrimation, piloerection, pupil size, and unusual respiratory pattern were observed. 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.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related body weight changes were noted. No significant differences were noted in absolute body weight change over the course of the study for either sex.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of males and females of all dose groups was similar to the control group during the whole study. Food consumption of recovery treated rats was similar in comparison with control groups during the whole application and recovery period.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related changes in haematology or coagulation were noted.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant changes against normal physiological conditions were detected. In the urine of some animals, small amounts of protein and presence of blood (erythrocytes) were observed. There are no differences between control and the dose groups. These findings considered to be normal. No test item related effect was observed.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weight main difference was considered to be associated with administration of the test item is the increase in relative weights of liver after the treatment period 28 days in males and females and also after 14 days recovery period. The increase of relative liver weight was more pronounced in females than males. The higher relative liver weights are considered adaptive and hence non-adverse.
Description (incidence and severity):
Red discoloration of jejunum, thymus, stomach and mesenterial lymph nodes were most often occurring in rats treated with 500 mg/kg and 1000 mg/kg of test item. Frequent incidences of jejunum discoloration red or orange, diffuse or multifocal occurred in males: 4/7 (500 mg/kg), 3/7 (1000 mg/kg) and females: 2/7 (100 mg/kg) and 1/7 (1000 mg/kg). It occurred also in controls 1/7 in males and 1/7 in females. Frequent incidences of stomach discoloration occurred in 3/7 males of the high dose group (1000 mg/kg).
These discoloration-macroscopic findings are not considered to by test item related.
The summary of other observations included defect of kidney tissue in one rat treated with 1000mg/kg (♂127) and two rats treated with 500 mg/kg (♂120, ♂118); red fluid in urinary bladder in one Control male; urinary bladder discoloration in one male (100 mg/kg); hernia in liver in one female treated with 1000mg/kg (♀166) liver.
Observations at recovery sacrifice included following:
The incidence of discoloration prostate and jejunum (♂132) and incidence of kidney tissues defects in 2/5 (Control) and 1/5 (1000 mg/kg) females.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Histopathological changes in examined organs; 28-day oral treatment with 1000 mg/kg CR 1-(9,9-Dibutyl-9H-fluoren-2-yl)-2-methyl-2-morpholin-4-yl-propan-1-one did not cause any primary toxicity in Wistar rats. The increased cytoplasmic, mainly perilobular vacuolation in liver is supposed to be the physiological response of the organism, which is reversible after removal of applicated test item.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Considering results of this study following conclusions were made that the 28-day treatment
- All the male and female animals from control and all the treated dose groups survived throughout the dosing period of 28 days and the recovery period of 14 days
- No signs of intoxication were observed in male and female animals during the dosing period of 28 days and the recovery period of 14 days
- Male and female animals from the treated dose groups exhibited comparable body weight gain with that of controls after the 28-day treatment and 14-day recovery period
- Food consumption of control and treated animals was found to be comparable throughout the dosing period and the recovery period of 14 days
- Haematology a clinical chemistry analysis conducted at the end of the dosing period and at the end of recovery period revealed no abnormalities attributable to the treatment
- Functional battery observation tests conducted at the end of the dosing period and at the end of recovery period revealed no abnormalities
- No histological consequence or no toxicological relevance was attributed to the macroscopically found changes in the gross pathological investigation.
- Organ weight data was found to be comparable with that of respective controls except of liver; the increase in relative weights of liver after the treatment period 28 days in males and females as well as after 14 days recovery period was considered to be associated with administration of the test substance.
- Histopathological changes in examined organs; 28-day oral treatment with 1000 mg/kg CR 1-(9,9-Dibutyl-9H-fluoren-2-yl)-2-methyl-2-morpholin-4-yl-propan-1-one did not cause any primary toxicity in Wistar rats. The increased cytoplasmic, mainly perilobular vacuolation in liver is supposed to be the physiological response of the organism, which is reversible after removal of
applicated test item.
NOAEL (no-observed-adverse-effect level)
Based on the results of the present study the NOAEL of the test item 1-(9,9-Dibutyl-9H-fluoren-2-yl)-2-methyl-2-morpholin-4-yl-propan-1-one is 1000 mg/kg.