Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-07-17 to 2020-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ditetradecyl peroxydicarbonate
EC Number:
258-436-4
EC Name:
Ditetradecyl peroxydicarbonate
Cas Number:
53220-22-7
Molecular formula:
C30H58O6
IUPAC Name:
ditetradecyl peroxydicarbonate
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for toxicity studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: Male animals: 41 – 43 days old Female animals: 36 – 38 days old
- Weight at study initiation: Male animals: 181 – 207 g Female animals: 119 – 144 g
- Fasting period before study: no
- Housing: 5 animals of the same sex/ cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30-70 %
- Air changes: > 10 per hr
- Photoperiod: 12/12 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
oral (gavage)
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of the Test Facility not longer than for three days before the administration and stored in the refrigerator.

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore PEG 400 was used for preparing formulations appropriate for oral administration. PEG 400 was a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: The test item was formulated in the vehicle in concentrations of 200 mg/mL, 60 mg/mL and 20 mg/mL.
- Amount of vehicle: final volume applied: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (control of concentration and homogeneity) were performed in the Analytical Laboratory of Test Facility three times during the study. Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on 3 occasions. Similarly, five samples were taken from the vehicle (Group 1) and analyzed.
Concentrations of the test item in the dosing formulations varied between ranges of 91 % and 93 % of nominal concentrations and formulations were homogeneous at each analytical occasion.
The suitability of the chosen vehicle for the test item at the intended concentrations was verified up front. Test item recovery was 105 and 99 % of nominal concentrations at 1 and 200 mg/mL in PEG 400, respectively. The test item proved to be stable in PEG 400 at room temperature for 24 hours (recovery was 110 % of starting concentration at 1 mg/mL and 107 % at 200 mg/mL) and at 5 ± 3°C for 3 days (recovery was 110 % of starting concentration at 1 mg/mL and 107 % at 200 mg/mL).
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
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:
control and high dose groups:
10 animals/sex
+ 5 animals/sex as satellite groupe for recovery investigations

Mid and low dose:
10 animals/sex
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: The dose setting with 100, 300 and 1000 mg/kg bw/day is based on findings obtained in a previous repeated dose toxicity study according to OECD 422 with the test item in the rat. Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for animal assignment: All animals were sorted according to body weight and divided to weight groups aided by a computerized calculation. There was an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean weight of animals from all test groups were as uniformly as practicable. Grouping was aided by SPSS/PC+ software, verifying the homogeneity and variability between the groups and cages according to the actual body weight.
- Fasting period before blood sampling for clinical biochemistry: 16 hours
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to first exposure, then once weekly
- parameters examined:
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 (treatment and recovery periods).

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, then twice weekly for four weeks (weeks 1-4) then once weekly (weeks 5-13) during the course of the treatment period and once weekly during the recovery period (weeks 13-17).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- reweighing of food in weekly intervals

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimatisation period and at test termination (Day 89)
- Dose groups that were examined: control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last treatment or at the end of recovery period (at necropsy)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after last treatment or at the end of recovery period (at necropsy)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of exposure (Days 82)
- Dose groups that were examined: all animals of control and high dose; due to no effects observed mid and low dose animals were not examined
- Battery of functions tested: sensory activity / grip strength / motor activity


IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes; full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups including recovery groups.
Other examinations:
Determination of Serum Levels of Thyroid Hormones
Blood samples for measurements of thyroid hormones were collected as it is described in paragraph “Clinical Chemistry”. The quantitative determination of circulating FT3, FT4 and TSH in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immuno-chemistry analyzer.

Examination of Estrous Cycle
The estrous cycle of all female animals was examined during the last two weeks of the treatment period (from Day 77 up to and including Day 90 or 91, depending on the day of the necropsy). A vaginal smear was prepared from each female. After drying, smears were stained with 1 % aqueous methylene blue solution and were examined by a light microscope. Type of cycle (regular or irregular), cycle length, number of cycles during the two weeks, number of days in pro-estrous, estrous and diestrous, number of animals with prolonged diestrous, number of animals with prolonged estrous were evaluated.
Statistics:
Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- estrous cycle
- hematology
- blood coagulation
- clinical chemistry
- organ weight data
- thyroid hormones
- sperm parameters
The heterogeneity 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.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No adverse signs of systemic toxicity related to the test item were detected at any dose level (100, 300 or 1000 mg/kg bw/day) at the daily clinical observations.

Treatment period
The behavior and physical condition of control male animals were considered to be normal at 100, 300 or 1000 mg/kg bw/day during the entire treatment period.
Salivation was observed in male (15/15) and in female (13/15) animals administered with 1000 mg/kg bw/day with variable frequency and onset shortly after the daily treatment and with short duration from Day 28 up to the termination of the treatment period. Based on the short time frame of observation, this finding was judged to be test item related but not adverse. Animals in the control and lower dose groups did not salivate therefore salivation was considered to be related to the treatment with the high dose of the test item.
Softer than normal stool was seen in the bedding material in each cages of the control, 100, 300 and 1000 mg/kg bw/day groups during the observation period from Day 4 (male and female). The slight change in the consistency of the feces was also observed in control animals, therefore, it was considered to be related to the vehicle and not adverse as no related findings were detected at the examined parameters.

Recovery period
Clinical signs were not detected in animals of the control or 1000 mg/kg bw/day (male or female) dose group during the recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two female animals – 1/10 at 100 mg/kg bw/day and 1/10 at 300 mg/kg bw/day – were found dead on Day 19. There were no preceding clinical signs referring to weak condition of these animals. Determining the cause of death was not feasible because the vital organs were cannibalized after the death.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was unaffected by the test item at 100, 300 and 1000 mg/kg bw/day (male and female animals) during the 3-month treatment period.

Treatment period
The mean body weight was similar to their control in male and female animals treated with 100, 300 or 1000 mg/kg bw/day during the entire treatment period.
Some statistically significant difference with respect to the control was detected at the mean body weight gain in male animals at 100 mg/kg bw/day between Days 84 and 89 (higher), at 300 mg/kg bw/day between Days 28 and 35 (higher) and at 1000 mg/kg bw/day between Days 70-77 (lower) and between Days 84 and 89 (higher). These minor changes in the body weight gain had no influence on the mean body weight or on the summarized body weight gain and therefore had no toxicological relevance.
The mean body weight and body weight gain were comparable – i.e. there were no statistically significant differences – in the control and test item treated female animals (100, 300 and 1000 mg/kg bw/day) during the entire treatment period.

Recovery period
The mean body weight and body weight gain was similar to the control in male animals at 1000 mg/kg bw/day during the recovery period.
The mean summarized body weight gain was slightly lower than in the control group in female animals at 1000 mg/kg bw/day during the post-treatment observation period (between Days 90-117). However, the mean body weight of these female animals was similar to the mean body weight of the control animals, therefore the slight changes in the mean body weight gain were toxicologically not relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no test item influence on the mean daily food consumption of male or female animals at 100, 300 or 1000 mg/kg bw/day during the treatment period.

Treatment period
Statistically significant differences with respect to the control were observed for the slightly higher mean daily food consumption in male animals as follows:
- at 100 mg/kg bw/day on week 1;
- at 300 mg/kg bw/day on weeks 1, 6, 9 and 10;
- at 1000 mg/kg bw/day on weeks 1 and 6;
The mean daily food consumption was comparable with the control in female animals at 100, 300 and 1000 mg/kg bw/day during the entire treatment period.
The minor differences were considered to be indicative of the biological variation and not related to the test item in the mean food consumption of male animals. The values met well the historical control values and there was no dose relevance.

Recovery period
During the course of the recovery period, the mean daily food consumption of male animals was similar in the control and high dose group.
In the female animals at 1000 mg/kg bw/day, the mean daily food consumption slightly exceeded the control value during the second and third weeks of the post-treatment period. This slight difference was considered to be toxicologically not relevant as similar findings were not detected during the course of the treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The eyes were without any abnormalities in all animals before the treatment and in animals in the control and high dose groups at termination of the treatment.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (100, 300 or 1000 mg/kg bw/day).

Treatment period
The examined hematological and blood coagulation parameters were similar to the control in male animals at 100 mg/kg bw/day.
Compared to the respective control group, slight reductions were detected in the mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), mean platelet count (PLT) and in mean prothrombin time (PT) in male animals at 300 mg/kg bw/day and in the mean platelet count at 1000 mg/kg bw/day.
In the female animals, the mean prothrombin time was slightly but statistically significantly shorter than in the control group at 100 and 1000 mg/kg bw/day.
All examined hematological and blood coagulation parameters were comparable to the respective value in the control group in female animals at 300 mg/kg bw/day.

Recovery period
The mean red blood cell count (RBC) and hematocrit value (HCT) were slightly lower than in the control group in male animals in 1000 mg/kg bw/day group at the end of the post-treatment period.
In the female animals in 1000 mg/kg bw/day recovery group, statistically significant changes were revealed with respect to the control group at the slightly lower mean percentage of neutrophil granulocytes (NEU), at the slightly higher mean percentage of lymphocytes (LYM) and at the lower mean hematocrit (HCT).
The slight changes in hematology or blood coagulation parameters were judged to have no toxicological relevance as these were with minor degree, not related to doses and values met well historical control (MCH, MCHC, PLT, PT in male animals and NEU, LYM, HCT and PT in female animals).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse test item effect was detected at the evaluation of clinical chemistry parameters for all dose groups (100, 300 and 1000 mg/kg bw/day).

Treatment period
In the male animals treated with 100 mg/kg bw/day, elevated mean concentration of inorganic phosphorous (Pi) and lower mean concentration of low-density lipoproteins (LDL) were revealed in comparison with the control group.
The mean glucose concentration (GLUC) was slightly higher and the mean concentrations mean low-density lipoproteins and potassium (K+) were lower than in the control group in male animals at 300 mg/kg bw/day.
Statistically significant difference with respect to the control was detected at the lower mean concentrations of urea, blood urea nitrogen (BUN), cholesterol (CHOL), high-density lipoproteins (HDL), low-density lipoproteins and potassium and the slightly higher albumin: globulin ratio (A/G) in male animals at 1000 mg/kg bw/day.
The examined clinical chemistry parameters were comparable in the female animals in control and 100 and 300 mg/kg bw/day groups.
The mean activity of aspartate aminotransferase (AST) was higher and the mean concentrations of urea and BUN was lower with respect to the control in the female animals at 1000 mg/kg bw/day groups.

Recovery period
Statistical significance with respect to the control was observed at the slightly lower mean concentrations of urea and blood urea nitrogen in male animals in 1000 mg/kg bw/day group at the end of the recovery period.
In the female animals, the examined clinical chemistry parameters were comparable in the control and 1000 mg/kg bw/day groups at the termination of the recovery period.
The sporadic statistical differences detected at some parameters were considered to be of little or no biological relevance. All these changes were with minor degree and not related to doses (GLUC, Pi, LDL, K+). Slight changes in AST, UREA, BUN, CHOL, HDL and A/G in animals at 1000 mg/kg bw/day were judged to be toxicologically not significant as there were no supporting findings (hematology, histopathology, related biochemical parameters).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery
The functional observation battery did not demonstrate any test item related differences with respect to the controls in the behavior or in reactions to different type of stimuli at 100, 300 or 1000 mg/kg bw/day (male and female). The behavior and reactions to different type of stimuli or manipulations of animals were judged to be normal in the control and all test item treated groups at the end of the treatment period (on Day 82). Softer than normal stool was also detected at the cage-side examinations in each cage (control, 100, 300 and 1000 mg/kg bw/day).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Test item related changes were not detected in the examined organ weights in male or female animals at 100, 300 or 1000 mg/kg bw/day.

Treatment period
Statistically significant differences with respect to the control were noted for the slightly higher mean epididymides weights (absolute and relative to body weight) in male animals in 1000 mg/kg bw/day dose groups at termination of the treatment period.
In the female animals, the mean weights of the kidneys were slightly lower than in the control group at 1000 mg/kg bw/day.

Recovery period
The weights of the examined organs were similar in the male animals in control and 1000 mg/kg bw/day groups at the end of the recovery period.
In the female animals at 1000 mg/kg bw/day, statistical significances were revealed for the higher mean liver weights (absolute and relative to body and brain weights) and higher mean spleen weights (absolute and relative to body and brain weights) as well as for the slightly higher mean adrenal gland weight relative to brain weight. All these statistically significant differences during treatment and recovery period, with respect to the appropriate control, were of small degree and no related findings were detected at the histopathological examinations. Moreover, the weights of these organs met well – i.e. were well within or marginal to – the historical control in the female animals. The weights of epididymides also were within the historical control range except for four animals at 1000 mg/kg bw/day. However, in the lack of morphological lesions, these minor changes in the epididymides weights had no biological meanings. Therefore, these changes in the organ weights were not considered to be toxicologically relevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period.

Treatment period
In dead female animal at 100 mg/kg bw/day (1/10), the organs and tissues were cannibalized – except for head (brain, pituitary, eyes, Harderian gland) and spinal column with spinal cord. Therefore, macroscopic observations were not feasible. In dead female animal at 300 mg/kg bw/day (1/10), the heart, abdominal aorta, sternum, skin and mammary gland, kidneys, adrenal glands, urinary bladder, ovaries and uterus with vagina were cannibalized. Remaining organs and tissues were slightly autolyzed and did not show macroscopic changes. In the male animals, one or both sided pyelectasia was observed in each group at the terminal necropsy as follows: 1/10 control; 4/10 at 100 mg/kg bw/day; 2/10 at 300 mg/kg bw/day; 5/10 at 1000 mg/kg bw/day. Lack of seminal fluid was observed in one side of seminal vesicle in one control male animal (1/10). In the surviving female animals, one or both sided pyelectasia (2/10 control) and moderate or marked hydrometra – 1/10 control, 1/9 at 100 mg/kg bw/day, 2/9 at 300 mg/kg bw/day and 5/10 at 1000 mg/kg bw/day – were detected at the terminal necropsy.

Recovery period
In the male animals, both sided pyelectasia was observed in single animals in control (1/5) and in one high dose (1/5) group at the end of the recovery period.
One side pyelectasia (1/5) and moderate or marked hydrometra (2/5) were seen in the control female animals at the termination of the post-treatment observation period. The organs and tissues were judged to be normal in the female animals in 1000 mg/kg bw/day recovery group. These findings are common in untreated experimental rats and were not related to the test item. Lack of seminal fluid was only detected in one control male animal. Hydrometra is indicative of female sexual cycle. Histological findings were in accordance with macroscopic observation in the uterus. Pyelectasia is a common finding in experimental rats of this strain with similar age. Pyelectasia occurred in each group of male animals independently from doses. There were no pathological alterations in the uterus or kidneys at the histopathological examination, therefore these were considered toxicologically not relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological examination did not reveal test item related adverse lesions in the organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period.
In control group, alveolar emphysema in the lungs (1/10 male and 2/10 female) occurred sporadically and was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination. Hyperplasia of bronchus associated lymphoid tissue (BALT) was observed in male animals terminally (1/10 control; 1/10 male at 1000 mg/kg bw/day) and in male and female animals at the end of the recovery period (1/5 control male, 2/5 male and 1/5 female at 1000 mg/kg bw/day). BALT is a physiological immunomorphological phenomenon, without toxicological significance. One or both sided pyelectasia was observed in the kidneys in several animals at termination of the treatment (1/10 male and 2/10 female control; 4/4 at 100 mg/kg bw/day; 2/2 at 300 mg/kg bw/day; 5/10 at 1000 mg/kg bw/day) and at the end of the recovery period (1/5 male and 1/5 female control, 1/5 male at 1000 mg/kg bw/day).
Pyelectasia without other histopathological lesions (degeneration, inflammation, fibrosis etc.) is a common slight individual disorder in laboratory rats, without toxicological significance. Histological examination revealed chronic progressive nephropathy (CPN) in two male animals (1/10 control and 1/10 at 1000 mg/kg bw/day) in minimal degree. CPN is an important spontaneous renal disease of the commonly used strains of laboratory rat. In one control male animal, decreased amount of secrete was observed in the seminal vesicle (one side, 1/10), which was considered as a slight functional disorder, without toxicological significance. The dilatation of the uterine horns in the female animals (1/10 control, 5/10 at 1000 mg/kg bw/day at termination and 2/5 control at the end of the recovery period) is a slight neuro-hormonal phenomenon in connection with the sexual function – pro-estrus phase – of the inner genital organs.
Alveolar emphysema in connection with the hypoxia, dyspnea and circulatory disturbance, developed during the exsanguination procedure occurred with similar incidence in the control and treated animals.
Hyperplasia of bronchus associated lymphoid tissue – a physiological immunomorphological phenomenon – was without toxicological significance and was with similar incidence in control and high dose male animals and was only present in control female but not in the high dose females.
Dilatation of uterine horn related to hydrometra is a slight neuro-hormonal phenomenon in connection with the sexual function – pro-estrous phase – of the inner genital organs. No morphological evidence of acute or subacute test item related injury (degeneration, inflammation, necrosis etc.) of the gastrointestinal tract, the liver, the pancreas, the respiratory system, the cardiovascular system, the urinary system, the lymphoid system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system, the eyes, the lachrymal glands, and the integumentary system, was observed. The structure and the cell morphology of the endocrine glands was the same in the control and treated animals.

Sperm Examinations
The sperm count, morphology and motility of sperm cells were not affected after three months administration of 1000 mg/kg bw/day of the test item.
The mean sperm count, percentage of motile cells and cells with normal morphology was similar in animals of the control and 1000 mg/kg bw/day groups at the termination of dosing period.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Clinical Biochemistry – Serum Levels of Thyroid Hormones
FT3, FT4 and TSH levels were not affected by the test item at 100, 300 or 1000 mg/kg bw/day (male and female) at the end of the treatment.
There were no significant differences with respect to the control in FT3, FT4 or TSH levels at 100 and 1000 mg/kg bw/day (male and female). The FT3 concentration slightly exceeded the control value in male animals at 300 mg/kg bw/day.
Statistical significance with respect to the control was observed in the male animals at 1000 mg/kg bw/day at the slightly higher FT3 level at the end of the recovery period. These slight differences with respect to the control were considered to be indicative of the biological variation and not related to the test item because there were no changes in the animals in the high dose at termination the treatment. The TSH concentrations were below the detection limit in all animals (0, 100, 300 and 1000 mg/kg bw/day), so it was considered to be in the physiological range of this strain, sex and age, as reflected by the historical control data.

Examination of Estrous Cycle
The estrous cycle was similar in the female animals in the control and test item administered groups during the last two weeks of the study. There were no significant differences between the control and test item treated groups in the percentage of female animals with regular or irregular cycles, mean number of cycles or mean cycle length, mean number of days in proestrous, estrous and diestrous or percentage of animals in prolonged diestrous.

Effect levels

Key result
Dose descriptor:
NOAEL
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 at the highest dose tested (limit dose)

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Please refer to result tables attached.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this OECD 408 compliant study the test item did not cause adverse effects in male or female Han: WIST rats after consecutive 90/91-day oral (by gavage) administration at 100, 300 or 1000 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL: 1000 mg/kg bw/day for male and female Han: WIST rats.
Executive summary:

The purpose of this study was to obtain information on the possible health hazards likely to arise from repeated exposure with the test item at three dose levels over a prolonged period of time (90 days) followed by four weeks recovery period in order to assess persistence or delayed occurrence of potential toxicological effects.

The test item was administered orally (by gavage) to male and female Han: WIST rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 or 91 days. Five animals/ sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations). The suitability of the chosen vehicle – Polyethylene glycol 400 – for the test item was analytically proven up front. Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted at the end of the treatment. The body weight was determined twice weekly for four weeks (weeks 1-4) then once weekly during the course of the treatment and recovery periods. Food consumption was measured and evaluated weekly. The estrous cycle of each female animal was examined for two weeks before necropsy. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in male animals in the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. Additionally, organs showing macroscopic changes were processed and evaluated histologically in the low and mid dose groups.

The results of the study were interpreted comparing test item treated groups with controls, which were administered concurrently with vehicle only.

There was no test item related mortality at any dose level (1000, 300 or 100 mg/kg bw/day). Two female animals (1/10 at 100 mg/kg bw/day and 1/10 at 300 mg/kg bw/day) were found dead for unknown reasons on Day 19.

No signs of toxicity related to the test item were detected at any dose level (100, 300 or 1000 mg/kg bw/day) at the daily or detailed weekly clinical observations or during the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. Salivation noted for male and female animals at 1000 mg/kg bw/day with different incidence during the treatment period was judged to be not adverse as animals salivated shortly after the daily administration of the test item for a short time period. Softer than normal stool in the bedding material was considered independent from the test item because it was seen in each cage in the control, 100, 300 and 1000 mg/kg bw/day groups from Day 4 up to the termination of the treatment. There were no clinical signs during the recovery period. The body weight development was unaffected by the test item at 100, 300 and 1000 mg/kg bw/day (male and female animals) during the 3-month treatment period. The mean body weight was similar to the control in male and female animals at 1000 mg/kg bw/day during the recovery period. The mean daily food consumption was comparable in animals of the control and test item treated groups (100, 300 and 1000 mg/kg bw/day).

There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day). A test item related influence on the estrous cycle was not detected (100, 300 and 1000 mg/kg bw/day).

Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (100, 300 and 1000 mg/kg bw/day). No pathologic test item effect was detected at the evaluation of clinical chemistry parameters (100, 300 and 1000 mg/kg bw/day). FT3, FT4 and TSH levels were comparable in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) at the end of the treatment and recovery periods. The concentrations of TSH were below the detection limit in all animals (0, 100, 300 and 1000 mg/kg bw/day) in accordance with the historical control data, so it was considered to be in the physiological range of this strain, sex and age. Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period. Sperm analysis did not reveal any test item related influence on the sperm parameters (count, motility and morphology) at 100, 300 or 1000 mg/kg bw/day. Test item related changes were not detected in the examined organ weights in male or female animals at 100, 300 or 1000 mg/kg bw/day. Histological examination did not reveal severe test item related lesions in the organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period.

In conclusion, based on results of the study, NOAEL was determined to be 1000 mg/kg bw/d.