N-[3-({[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}methyl)-3,5,5-trimethylcyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The test item was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422 and was shown to not adversely influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats. Based on these observations the No Observed Adverse Effect Levels (NOAEL) for reproductive performance of male/female rats was 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on 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:

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

Details on study schedule:

- Age at mating of the mated animals in the study: about 14 weeks

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

Parental animals: Observations and examinations:

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).

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Percentage of pairings, percentage of fertile pairings, percentage of infertile pairings, male copulatory index, male fertility index were calculated.
Testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands were preserved.
Testis weight and epididymis weight of all parental animals were determined.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
sex ratio, survival index of pups on postnatal day 4, Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4, Mean body weight gain per litter between postnatal days 0-4, Litter weight on postnatal days 0 and 4.
Lung flotation test was performed on offspring found dead on postnatal day 0.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals one day after the last treatment.
- Maternal animals: All surviving animals one day after the last treatment.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Tables 1 and 2 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
Offspring euthanized on day 4 post-partum, were carefully examined for gross abnormalities externally.

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examinations including the cervical, thoracic, and abdominal viscera, a lung flotation test was performed.

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 (i.e. control value).

Reproductive indices:

Copulatory Indices, Fertility Indices, Gestation Indices were determined.

Offspring viability indices:

Pre-implantation mortality, Post-implantation mortality, Intra uterine mortality, Post-natal mortality, Sex ratio and Survival Index were calculated.

Clinical signs:

no effects observed

Description (incidence and severity):

There was no test item related mortality in parental animals during the course of study at any dose level. Adverse signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No test item related mortality was observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was not affected.

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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):

The test item 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

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

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.

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 at 1000, 300 or 100 mg/kg bw/day in male or female animals.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Key result

Critical effects observed:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

The percentage of cold offspring was slightly higher than in the control group at 1000 mg/kg bw/day on postnatal day 0. However, this finding was not considered to be toxicologically relevant as it was transient and of low incidence. Moreover, this observation was not associated with any influence on the development of the offspring.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on pup’s mortality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A test item effect on the body weight development of the offspring was not found.

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:

no effects observed

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring.

Urinalysis findings:

not examined

Sexual maturation:

not examined

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

Description (incidence and severity):

Sex Distribution:
There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on postnatal days 0 or 4.

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 effect observed.

Key result

Critical effects observed:

not specified

Key result

Reproductive effects observed:

not specified

Conclusions:

The test item did not adversely influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats. The development of the F1 offspring from conception to day 4 post-partum, after repeated oral administration of the dams, was not impaired at any dose level. Based on these observations the No Observed Adverse Effect Levels (NOAEL) for reproductive performance of male/female rats and for F1 Offspring was 1000 mg/kg bw/day.

Executive summary:

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). All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. 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, there were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals. No adverse findings on offspring development (mortality, clinical signs, body weight) or at necropsy were detected in the offspring terminated as scheduled. Based on these findings, the NOAEL for reproductive performance of the parental animals and for the development of the offspring was determined to be 1000 mg/kg bw.

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:

well documented GLP and OECD 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

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422, 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). All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. 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. 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, there were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals. Based on these findings, the NOAEL for reproductive performance was determined to be 1000 mg/kg bw.

Effects on developmental toxicity

Description of key information

The test item was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422. The development of the F1 offspring from conception to day 4 post-partum, after repeated oral administration of the dams, was not impaired at any dose level. Based on these observations the No Observed Adverse Effect Levels (NOAEL) for F1 Offspring was 1000 mg/kg bw/day.

A reproduction/developmental toxicity screening test according to OECD guideline 422 wa sused for assessment of developmental toxicity and the effects on fertility.

Effect on developmental toxicity: 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:

well documented GLP and OECD Guideline Study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422, 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). All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. 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. 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. No adverse findings on offspring development (mortality, clinical signs, body weight) or at necropsy were detected in the offspring terminated as scheduled. Based on these findings, the NOAEL for the development of the offspring was determined to be 1000 mg/kg bw.

Justification for classification or non-classification

Based on results of the in vitro genetic toxicity studies (Ames test), SIKA Hardener MI was not classified for toxicity to reproduction according to Directive 67/548/EEC (DSD) and to Regulation (EC) No 1272/2008 (CLP).