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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2014-09-10 to 2014-11-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
[2,2-dimethyl-3-(morpholin-4-yl)propylidene][(5-{[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}-1,3,3-trimethylcyclohexyl)methyl]amine
EC Number:
700-584-3
Cas Number:
1217271-02-7
Molecular formula:
C28H52N4O2
IUPAC Name:
[2,2-dimethyl-3-(morpholin-4-yl)propylidene][(5-{[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}-1,3,3-trimethylcyclohexyl)methyl]amine

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation: Male animals: 83 – 87 days old, Female animals: 83 – 87 days old
- Weight at study initiation: Male animals: 330 – 399 g, Female animals: 205 – 252 g. The weight variation in animals involved at the starting point of the study did not exceed ± 20 % of the mean group weight of each sex.
- 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: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance, ad libitum
- Water: ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

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 beforehand, not longer than for 24 hours before use.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water, therefore, PEG 400 was used for preparing formulations appropriate for oral administration. PEG 400 was shown to be a suitable vehicle to facilitate formulation analysis for the test item. The same vehicle was used previously for very similar substances with good results. Sufficient historical control data with this vehicle are available.
- Concentration in vehicle: The test item was formulated in the vehicle in concentrations of 200, 60 and 20 mg/mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Recovery of the test item from PEG 400 formulations was measured using a reverse phase HPLC method with UV detection on a BDS Hypersil C18 column (150x4.6 mm 3 μm). Analysis of formulations (checking of concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility two times during the study. Five samples (5 mL, each) were taken from different places from each concentration (Groups 2, 3 and 4) and all samples were measured. Similarly, five samples (5 mL, each) were taken from the vehicle (Group 1), from different places, and analyzed.
Duration of treatment / exposure:
Male animals were dosed for 48 days, female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3-11 (altogether for 48 or 55 days depending on day of mating).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting based on findings obtained in a previous repeated dose oral gavage toxicity study with Sika Hardener MI in rats and after consultation of the Sponsor (Toxi-Coop study no. 644-400-0050). In general, the doses were selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Parent male animals were weighed on the first day of dosing (Day 0), weekly thereafter and on the day of necropsy.
Parent females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum day 0 (within 24 hours after parturition) and post-partum days 4 and 7, if it was feasible.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period except mating days (pre-mating and post mating for male animals and non-pregnant females, during pre-mating, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 7 for dams; latter, if was feasible).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 5 males and 5 females from each group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Animals fasted: Yes
- How many animals: 5 males and 5 females from each group
- Parameters checked in table 2 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes
Other examinations:
At the time of termination, body weight and weight of the testes, epididymides of all parental animals were determined with an accuracy of 0.01 g.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
Paired organs were weighed together; absolute organ weight was reported. Relative organ weight (to body and brain weight) were calculated and reported.
Statistics:
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.
The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group.

Results and discussion

Results of examinations

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.
Mortality:
no mortality observed
Description (incidence):
no mortality was observed at any dose level.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was not affected at 1000, 300 or 100 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item or treatment related changes in the food consumption of male and female animals at any dose level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item related toxic changes in the examined hematological parameters in male or female animals at any dose level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item related toxic alterations occurred in the examined clinical chemistry parameters.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The absolute and relative weights of examined organs did not demonstrate any test item related alterations.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No specific macroscopic alterations related to the test item effect were observed at the gross necropsy of animals.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The test substance did not cause any toxic or other test item related lesions detectable by histological examination in the genital and other organs of the experimental animals.
Histopathological findings: neoplastic:
no effects observed

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 were observed.

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, the test item administered orally (by gavage) at 1000, 300 or 100 mg/kg bw/day did not cause signs of systemic toxicity. The NOAEL was thus determined to be 1000 mg/kg bw.
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 substance 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 4 post-partum when repeatedly administered orally (by gavage) to parental animals at doses of 1000 mg/kg bw/day, 300 mg/kg bw/day and 100 mg/kg bw/day compared to control animals. Four groups of Hsd.Brl.Han: Wistar rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 1000, 300 and 100 mg/kg bw/day corresponding to concentrations of 0, 200, 60 and 20 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, Polyethylene glycol 400 (PEG 400). The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. The test item concentrations in the dosing formulations varied in the acceptable range between 90% and 102 % of the nominal values confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 48 days). Females were additionally exposed through the gestation period and up to lactation days 3 - 11, i.e. up to the day before necropsy (altogether for 48 or 55 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Each 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.

The dams were allowed to litter, and rear their offspring up to day 4 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 4. All parental animals were subjected to gross pathology one day after the last treatment. Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) and pituitary in the control and high dose groups. The reproductive organs and pituitary of non-pregnant females and cohabited males in the low and mid dose groups were also processed and evaluated histopathologically. Additionally, full histopathology was performed on the organs and tissues of animals selected for general toxicological examinations in the control and high dose groups.

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (Polyethylene glycol 400) only. Historical control data were also considered. As a result, no test item related signs of systemic toxicity were observed. Therefore, the NOAEL was determined to be 1000 mg/kg bw.