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

Description of key information

Under the conditions of the repeated dose oral toxicity study, the test item administered 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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

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

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.

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

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.

Key result

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 2014-05-18 to 2014-07-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Range-finding study following in general guideline procedures

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

14 day application, no histopathology

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

14 day application, no histopathology

Qualifier:

equivalent or similar to guideline

Guideline:

other: OPPTS 870.3050, July 2000

Deviations:

yes

Remarks:

14 day application, no histopathology

GLP compliance:

no

Remarks:

However the principles of GLP were followed and all data were recorded and retained.

Limit test:

no

Species:

rat

Strain:

Brown Norway

Remarks:

Hsd.Brl.Han: of Wistar origin

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: 47 – 52 days old
Female animals: 37 – 42 days old
- Weight at study initiation:
Male animals: 231 – 255 g
Female animals: 133 – 166 g
- Housing: Type III polypropylene/polycarbonate
- Bedding: Lignocel Hygienic Animal Bedding, supplier J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg, Germany).
- Diet: Animals received ssniff SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water: Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes (per hr): 10 - 15
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 pm

IN-LIFE DATES: From: 2014-05-18 To: 2014-07-02

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

REPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 200, 60 and 20 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility daily. Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility once during the study.

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 a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle: A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on day 0.
- Lot/batch no.: 14B110500

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) was performed in the Analytical Laboratory of Test Facility once during the study. The test item content was determined in PEG 400 formulations using the previously validated reverse phase HPLC method with UV detection on a BDS Hypersil C18 column (150x4.6 mm 3 μm).
Samples were taken on study Day 5 and measurement was conducted the same day (23 June 2014). The measured concentrations were between range of 87 and 98 % of the nominal concentrations of 200, 60 and 20 mg/mL.
The suitability of the chosen vehicle for the test item was analytically proven. Recovery was between 100 and 107 % of nominal concentrations at ~20 and ~200 mg/mL in Polyethylene glycol 400, respectively. Sika Hardener MI proved to be stable at room temperature for 4 hours and in a refrigerator (5 ± 3 °C) for two days.

Duration of treatment / exposure:

14 days

Frequency of treatment:

once a day

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Basis: nominal in PEG 400

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Basis: nominal in PEG 400

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Basis: nominal in PEG 400

No. of animals per sex per dose:

5 animals per dose per sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting at 1000, 300 and 100 mg/kg bw/day was based on the LD50 value of Sika Hardener MI and in agreement with the Sponsor. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day. Observations were performed on the skin, fur, eyes and mucousmembranes, 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.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed with an accuracy of 1 g on Days 0, 7 and 13. Individual body weight changes were calculated between Days 0-7, 7-13 and between Days 0-13, latter for the total body weight gain. Fasted body weight was measured on day of necropsy (Day 14).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: Once per week. The food consumption was determined with an accuracy of 1 g on Days 7 and 13 by reweighing the non-consumed diet.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of treatment (week 2)
- Anaesthetic used for blood collection: Yes, Isofluran anesthesia
- Animals fasted: Yes, about 16 h prior to blood collection
- How many animals: all animals
- Parameters: White Blood Cell (leukocyte) count, Red Blood Cell (erythrocyte) count, Hemoglobin concentration, Hematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Hemoglobin, Mean Corpuscular (erythrocyte) Hemoglobin Concentration, Platelet (thrombocyte) count, Reticulocytes, Differential white blood cell count, Activated partial Thromboplastin Time, Prothrombin Time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of treatment (week 4)
- Animals fasted: Yes, about 16 h prior to blood collection
- How many animals: all animals
- Parameters: Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Gamma Glutamyltransferase activity, Alkaline Phosphatase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Bile acids, Inorganic phosphate concentration, Calcium concentration, Sodium concentration, Potassium concentration, Chloride concentration, Total Protein concentration, , Albumin concentration, Albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Clinical Pathology
Clinical pathology examinations including hematology and clinical chemistry were conducted one day after the last treatment. Animals were food deprived for approximately 16 hours prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for determination of blood clotting times (for APTT and PT; 1.0 ml 9NC Greiner Bio-One International AG), one for hematology (MiniCollect® K3EDTA, 0.25 mL, Greiner Bio-One International AG), and the third one (Vacuette® Serum Tube, 2.5 ml, Greiner Bio-One International AG) to obtain serum samples for clinical chemistry.

Pathology:
Gross necropsy was performed on each animal. Animals were euthanized by exsanguination after verification of narcosis by Isoflurane CP® one day after the last treatment. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size.

Organ Weight:
The following organ weights were determined and recorded:
With precision of 0.01g: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands; With precision of 0.001g: adrenals, ovaries.

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- hematology
- clinical chemistry
- organ weight data
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. The frequency of clinical symptoms and pathologic findings were calculated.

Clinical signs:

no effects observed

Description (incidence and severity):

Test item or treatment related salivation was detected in male (4/5) and female (1/5) animals of 1000 mg/kg bw/day with different individual onset and incidence. The behavior and physical condition were considered to be normal in all male and female animals of 1000, 300, 100 mg/kg bw/day and control groups during the entire observation period.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in the control, 1000, 300 or 100 mg/kg bw/day groups during the course of 14-day treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight parameters were not affected during the entire observation period (1000, 300 or 100 mg/kg bw/day). There were no statistically or biologically significant differences in the mean body weight or body weight gain with respect to the control in male or female animals (1000, 300 and 100 mg/kg bw/day) during the course of the study.

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 (1000, 300 and 100 mg/kg bw/day). The mean daily food consumption was similar to that of the control group in male and female animals of 1000, 300 and 100 mg/kg bw/day groups during the 14-day observation period.

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):

Hematological examinations did not reveal adverse test item related changes in the investigated hematological parameters. Statistical significances were detected for male animals at the slightly less mean percentage of the basophil granulocytes (BASO) and at the slightly higher mean corpuscular hemoglobin content (MCH) at 1000 and 100 mg/kg bw/day. All these slight changes were judged to be of little or no biological significances. Values were well within the historical control ranges (BASO and MCH) and in case of all listed parameters or were not related to doses (MCH). In the female animals, statistical or biological significances were not detected with respect to the control in the examined parameters at 1000, 300 or 100 mg/kg bw/day.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item related adverse changes in the investigated clinical chemistry parameters of male or female animals at any dose level (1000, 300 and 100 mg/kg bw/day). Compared to the relevant control, statistically significant differences were observed at the higher mean activity of alanine aminotransferase (ALT) and less mean concentrations of sodium (Na+), chloride (Cl-) and albumin (ALB) in male animals administered with 1000 mg/kg bw/day dose. The mean sodium concentration was also less than in the control group in 300 mg/kg bw/day dose group of the male animals. In the female animals, slight but statistically significant differences with respect to the relevant control were observed at the higher mean albumin concentration at 100 mg/kg bw/day, at the less mean concentration of total protein (TPROT) at 1000 mg/kg bw/day and at the less ration of albumin:globulin (A/G) at 300 mg/kg bw/day. These sporadic statistical differences (ALT, Na+, Cl-, ALB, TPROT and A/G ration) were considered to be of little or no biological relevance. Although the mentioned differences between the control and test item treated groups were statistically significant were with small degree and all values remained well within or marginal to the historical control ranges for these parameters. Therefore these findings were not considered to be of toxicological relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Test item related adverse effects were not found on the weight of the examined organs in male or female animals (1000, 300 and 100 mg/kg bw/day). Slight statistically significant difference with respect to control was noted for male animals in the less mean liver weights relative to body weight at 1000 and 100 mg/kg bw/day and in the less mean kidney weights relative to body weight at 300 mg/kg bw/day, respectively. The difference with respect to the control was small and values remained well within the historical control range, moreover there were no related findings in the examined blood parameters or at the macroscopic examination of these organs. In the female animals, all examined organ weights (absolute and relative to body and brain weights) were similar to the relevant control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Test item related adverse macroscopic changes were not detected at the necropsy (1000, 300 or 100 mg/kg bw/day). Smaller than normal testes and epididymides were observed in one control male animal (1/5) and slight or moderate hydrometra was noted for single animals in each group (1/5 control, 1/5 at 1000 mg/kg bw/day, 1/5 at 300 mg/kg bw/day and 1/5 at 300 mg/kg bw/day). Changes of genital organs of male animals and hydrometra (latter indicative of sexual cycle of female animals) are frequent observations in experimental rats and were considered to be incidental individual alterations.

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed.

Key result

Critical effects observed:

not specified

Conclusions:

Under the conditions of the 14-day range-finding toxicity study, Sika Hardener MI did not cause adverse effects in male or female Hsd.Brl.Han:Wistar rats during the course of a consecutive 14 days oral administration at 1000, 300 or 100 mg/kg bw/day. The doses for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study) were determined as follows: 100, 300 and 1000 mg/kg bw/day.

Executive summary:

The aim of this 14 -day range-finding toxicity study was to obtain initial information on the toxic potential of Sika Hardener MI after sub-acute repeated application in rats at three dose levels and to allow dose-setting for a subsequent Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study). The test item was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats (n=5 animals/sex/group) once a day at 0 (vehicle control), 1000 300 and 100 mg/kg bw/day corresponding to concentrations of 200, 60 and 20 mg/mL. The application volume was 5 mL/kg bw for 14 days. Analytical control of dosing formulations was performed once during the study. The suitability of the chosen vehicle for the test item was analytically verified. Recovery was in the acceptable range between 100 and 107 % of nominal concentrations at ~20 and ~200 mg/mL in Polyethylene glycol 400 (PEG 400), respectively. Sika Hardener MI was proved to be stable at room temperature for 4 hours and in refrigerator (5 ± 3 °C) for 2 days. Measured concentrations of formulations applied in the study varied in the acceptable range between 87 and 98 % of the nominal concentrations and all formulations were homogenous, thereby confirming proper dosing. Detailed clinical observations were made on all animals once a day, after treatment at approximately the same time. Body weight and food consumption were measured weekly. Clinical pathology and gross pathology examinations were conducted at the end of the treatment period. Selected organs were weighed.

Results:

Mortality

No test item related mortality occurred at any dose level (1000, 300 and 100 mg/kg bw/day) during the observation period.

Clinical observations

Test item related salivation was observed in male or female animals dosed with 1000 mg/kg bw/day shortly after the administration for some minutes. The behavior and physical condition were considered to be normal in all male and female animals of 1000, 300, 100 mg/kg bw/day and control groups during the entire observation period.

Body weight and body weight gain

There were no test item-related adverse effects on the body weight parameters at 1000, 300 or 100 mg/kg bw/day doses.

Food consumption

The mean daily food consumption was similar to the control in male and female animals dosed with 1000, 300 or 100 mg/kg bw/day during the study.

Clinical pathology

There were no test item related changes in the examined hematological or clinical chemistry parameters in male or female animals administered with 1000, 300 or 100 mg/kg bw/day dose of the test item.

Organ pathology

Specific macroscopic alterations of the organs or tissues and changes in the investigated organ weight related to the test item were not detected.

Conclusion

Under the conditions of the present study, Sika Hardener MI did not cause adverse effects in male or female Hsd.Brl.Han:Wistar rats during the course of a consecutive 14 days oral administration at 1000, 300 or 100 mg/kg bw/day. No test item related adverse effects were observed at 1000, 300 or 100 mg/kg bw/day (male and female). 1000 mg/kg bw/day dose caused salivation in male and female animals.

Based on these observations the doses for a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study) were determined as follows:

Group 1 Vehicle control

Group 2 100 mg/kg bw/day

Group 3 300 mg/kg bw/day

Group 4 1000 mg/kg bw/day

Endpoint conclusion

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

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a 14 -day range-finding toxicity study Sika Hardener MI was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats (n=5 animals/sex/group) once a day at 0 (vehicle control), 1000, 300 and 100 mg/kg bw/day corresponding to concentrations of 200, 60 and 20 mg/mL. The application volume was 5 mL/kg bw for 14 days. Analytical control of dosing formulations was performed once during the study. The suitability of the chosen vehicle for the test item was analytically verified. Recovery was in the acceptable range between 100 and 107 % of nominal concentrations at ~20 and ~200 mg/mL in Polyethylene glycol 400 (PEG 400), respectively. Sika Hardener MI was proven to be stable at room temperature for 4 hours and in refrigerator (5 ± 3°C) for 2 days. Measured concentrations of formulations applied in the study varied in the acceptable range between 87 and 98 % of the nominal concentrations and all formulations were homogenous, thereby confirming proper dosing. Detailed clinical observations were made on all animals once a day, after treatment at approximately the same time. Body weight and food consumption were measured weekly. Clinical pathology and gross pathology examinations were conducted at the end of the treatment period. Selected organs were weighed.

Mortality

No test item related mortality occurred at any dose level (1000, 300 and 100 mg/kg bw/day) during the observation period.

Clinical observations

Test item related salivation was observed in male or female animals dosed with 1000 mg/kg bw/day shortly after the administration for some minutes. The behavior and physical condition were considered to be normal in all male and female animals of 1000, 300, 100 mg/kg bw/day and control groups during the entire observation period.

Body weight and body weight gain

There were no test item-related adverse effects on the body weight parameters at 1000, 300 or 100 mg/kg bw/day doses.

Food consumption

The mean daily food consumption was similar to the control in male and female animals dosed with 1000, 300 or 100 mg/kg bw/day during the study.

Clinical pathology

There were no test item related changes in the examined hematological or clinical chemistry parameters in male or female animals administered with 1000, 300 or 100 mg/kg bw/day dose of the test item.

Organ pathology

Specific macroscopic alterations of the organs or tissues and changes in the investigated organ weight related to the test item were not detected.

Under the conditions of the present study, Sika Hardener MI did not cause adverse effects in male or female Hsd.Brl.Han:Wistar rats during the course of a consecutive 14 days oral administration at 1000, 300 or 100 mg/kg bw/day. No test item related adverse effects were observed at 1000, 300 or 100 mg/kg bw/day (male and female). 1000 mg/kg bw/day dose caused salivation in male and female animals.

Main study:

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.

Justification for classification or non-classification

Based on the results of the available studies, Sika Hardener MI is not classified and labelled for long-term toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No. 2017/776.