Reaction product of propylidynetrimethanol, propoxylated, reaction products with ammonia and 2,2-Dimethyl-3-(4-morpholinyl)propanal

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-09-25 to 2015-05-15

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: EPA Health Effects Test Guidelines, OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000

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: Males: 14-15 weeks old, Females: 14-15 weeks old
- Weight at study initiation: Males: 352 - 422 g, Females: 204 - 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" 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: 34 days

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

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared in the formulation laboratory of the Test Facility daily. Analysis of formulations (checking of concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility two times during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 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.
- Lot/batch no.: 14B110500

Details on mating procedure:

- M/F ratio per cage: 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
- Length of cohabitation: Females were cohabited with the same male until copulation occurred or two weeks elapsed.
- Proof of pregnancy: Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422/OPPTS 870.3650). Sperm positive females were caged individually.
- Any other deviations from standard protocol: One control female animal (no. 125) failed to mate within 14 days therefore it was re-mated with other male of the control group, which has completed mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Checking of concentration of test item in formulations prepared in Polyethylene glycol 400. The test item was analyzed using HPLC with MS detection based on the peaks of oligomers of main constituent.

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. Date of sampling and analysis: October 01 and November 05, 2014.

Apparatus:

UHPLC system: Flexar LC Solvent Manager
No.: 260S12071605F
Flexar FX UHPLC Autosampler
No.: 293H2080102B
Flexar FX-10 UHPLC Pump A
No.: 291S12071663FX
Flexar Column Oven
No.: OVPF120715458

Mass spectrometer: AB SCIEX QTRAP 5500
No.: AU29511207

Balances: BL 120S Sartorius, No.: 15307011
AB54-S Mettler-Toledo, No.: 1122092721
BP 210D Sartorius, No.: 60602907

Vortex: Reamix 2789, No.: 730170

Ultrasonic bath: Branson 3210, No.: A700343D

HPLC Conditions

Column: Purospher STAR RP-18 endcapped 30-2 mm, 3 μm; No.: 720093
Mobil Phase: 1 mM Ammonium Formate in Methanol
Flow Rate: 0.6 mL/min
Injection volume: 5 μL
Run time: 5 minutes

MS Conditions

Scan Type: Q1 Multiple Ions (Q1 MS)
Polarity: positive
Ion Source: Heated Nebulizer
Ionisation mode Atmospheric Pressure Chemical Ionization (APCI)
Detector: Q1 Mass (Da): 824 for (x+y+z) = 4 oligomer Dwell: 200 msec
Q1 Mass (Da): 882 for (x+y+z) = 5 oligomer Dwell: 200 msec
Q1 Mass (Da): 940 for (x+y+z) = 6 oligomer Dwell: 200 msec
Parameter Table: Curtain Gas (CUR): 30 Temperature (TEM): 500 Nebulizer gas (GS1): 35 Heater Gas (GS2): 25 Nebulizer Current (NC): 4 DP (Declustering Potential): 150 EP (Entrance Potential): 10
Run time: 5 minutes

Duration of treatment / exposure:

Male animals were dosed for 43 days, female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3-6 (altogether for 43 - 58 days depending on day of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum.

Frequency of treatment:

Daily

Details on study schedule:

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

Dose / conc.:

0 mg/kg bw/day (actual dose received)

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:

12 males and 12 females per group

Control animals:

yes

Details on study design:

The dose setting with 1000, 300 and 100 mg/kg bw/day doses based on findings obtained in a previous repeated dose oral gavage toxicity study with Sika Hardener MTJ in rats and after consultation of the Sponsor (Toxi-Coop study no. 644-400-0049). 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: Males: First day of dosing, weekly thereafter and on the day of necropsy. Females: Weighed on the first day of dosing, then weekly on gestation days 0, 7, 14 and 21 and on post-partum day 0 and post-partum day 4. Additionally, female animals were weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight was measured on day of necropsy for animals subjected to organ weighing (all male animals and females selected for further examinations).

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 and 4)

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
- 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.

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.

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
Detailed histological examinations were performed on the ovaries, uterus, vagina, pituitary, testes and epididymides of all animals in the control and high dose groups.
Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
At the time of termination, fasted body weight (all male animals) 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. Fasted body weight of these female animals was determined, too. Paired organs were weighed together; absolute organ weight was reported. Relative organ weight (to body and brain weight) were calculated and reported.

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 statistical evaluation of appropriate data (marked with † above) were performed with the statistical program package SPSS PC+4.0.
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.

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

Mortality:

mortality observed, treatment-related

Description (incidence):

Test item related death occurred at 1000 mg/kg bw/day (3/12 males and 4/12 females). Three male animals died on Days 26, 34 or 40. Three pregnant animals and one dam were found dead on gestation days 21, 22 or on lactation day 3.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was slightly reduced at 1000 mg/kg bw/day (male and female). The body weight gain of male animals at 1000 mg/kg bw/day was reduced during the first two weeks (pre-mating period) of the study. This resulted in a statistically significant difference in the total body weight gain and in the mean body weight compared to the control from Day 13 up to the end of the study. As these changes was associated with a slightly less mean body weight of these animals (approximately -5 % or -6%), this alteration therefore was not considered to be toxicologically significant.
The body weight gain exceeded that of the control group in male animals at 300 and 100 mg/kg bw/day between Days 34 and 41. However, these changes did not cause relevant or significant changes in the mean body weight of these groups.
The body weight development was similar to that of the controls for the female animals administered with 1000, 300 or 100 mg/kg bw/day of Sika Hardener MTJ during the pre-mating and gestation periods. Slight statistical significance was only detected for the higher mean body weight gain of female animals at 300 mg/kg bw/day between Days 0 and 7, which had no influence on the mean body weight and was judged to be indicative of biological variation and not related to the test item. The mean body weight of dams at 1000 mg/kg bw/day was slightly but statistically significantly less than in the control group on lactation days 0 and 4.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The test item caused hyaline droplet nephropathy and tubular necrosis in the kidneys of prematurely died animals, and hyaline droplet nephropathy in the kidneys of male animals at 1000 mg/kg bw/day.
Histopathological examination revealed hyaline droplet nephropathy and tubular necrosis in the kidneys and congestion and alveolar edema in the lungs in all dead animals (3/3 male and 4/4 female). These lesions were considered as related to the application of the high dose of the test item. In one of the prematurely died female animal hemorrhage was detected in the uterus, which was considered as individual disorder after the delivery.
Hyaline droplet nephropathy was also observed in the kidneys of male animals (5/5) at 1000 mg/kg bw/day randomly selected for histopathological examination. Hyaline droplet nephropathy and “hydrocarbon nephropathy” are terms describe a spectrum of morphologic changes in the kidneys of male rats induced by a variety of structurally related and unrelated compounds. There is abnormal accumulation of a-2µ-globulin phagolysosomes of tubular epithelium. One proposed mechanism suggests that the chemical or a metabolite binds with the a-2µ-globulin or alters the structure so that the tubular cell lysosomal enzymes cannot degrade the protein complex. Other proposed mechanisms include a direct cytotoxic effect. It is unlikely that the various chemicals associated with hyaline droplet nephropathy in the male rat act by the same mechanism. Some chemicals which produce hyaline droplet nephropathy in male rats produce renal toxicity (unassociated with a-2µ-globulin) in female rats, whereas other chemicals produce no effects in the kidney of female rats. Marked tubular necrosis and regeneration of tubular epithelium are sometimes seen.
No hyaline droplet nephropathy or tubular necrosis in the kidneys was seen in the surviving female animals (5/5) of the high dose group or in male (5/5) and female animals (5/5) of the mid dose group (300 mg/kg bw/day) randomly selected for histopathological examinations.
Alveolar emphysema in minimal or mild degree was observed in the lungs of male (1/5 control and 2/5 at 1000 mg/kg bw/day) and female (2/5 control) animals. Pulmonary emphysema occurred sporadically and was considered to be consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in some control and test item treated animals (1/5 male and 1/5 female control; 1/5 male and 1/5 female at 1000 mg/kg bw/day) is a physiological phenomenon.
Dilatation of the uterine horns in non-pregnant female animal (1/12) at 1000 mg/kg bw/day is a slight neuro-hormonal phenomenon in connection with the sexual function – estrus phase – of the inner genital organs.

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

The investigated organs of reproductive system (testes, epididymides), were histologically normal and characteristic on the sexually mature organism in all examined male animals at 1000 mg/kg bw/day (12/12) and in the control group (12/12 for both groups). 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. The histological picture of epididymides and pituitary was normal in all cases as well.
The ovaries had a normal structure characteristic on the species, age and phase of the active sexual cycle in all investigated female animals (dams and non-pregnant female) in the control and test item treated groups (12/12 for both groups). The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well. The uterus, cervix, vagina and pituitary had a normal structure in accordance with the phase of sexual cycle in the investigated animals.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the digestive system, cardiovascular system, immune system, hematopoietic system, the skeleton, the muscular system, the male reproductive system or the central, or peripheral nervous system was detected.
No difference was observed by light microscopic investigation regarding the quantity or cyto-morphology of lymphoid cells in the spleen, thymus, lymph nodes, Peyer’s patches and of the myeloid and erythroid blast cells in the bone marrow (in the sternum and femur) between the test items treated and control animals.
The structure and the cell morphology of the endocrine glands was the same in the control and treated 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: NOAEL for reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Body weight
The offspring’s body weight development was slightly depressed between postnatal days 0 and 4.
Statistically significant difference with respect to the control was detected at 1000 mg/kg bw/day in the slightly lower mean litter weight on postnatal day 4 and at the lower mean litter weight gain between postnatal days 0 and 4.
There were no statistically significant differences between the control and test item treated groups in the body weight and body weight gain of offspring however the mean values remained below that of the control group on postnatal days 0 and 4, as well as between postnatal days 0 and 4.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL for F1 Offspring

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of the present study, Sika Hardener MTJ administered orally (by gavage) at 1000 mg/kg bw/day caused premature death and hyaline droplet nephropathy and tubular necrosis in the kidneys in male (3/12) and female (4/12) Hsd.Brl.Han: Wistar rats. Slightly reduced body weight development, food consumption, increases in the kidneys weights (males and females) and hyaline droplet nephropathy (males) were observed in surviving animals at 1000 mg/kg bw/day. There was no adverse influence on the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats at 1000 mg/kg bw/day. At 300 and 100 mg/kg bw/day, there were no test item related adverse alterations or impairment of the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female animals. The offspring’s body weight development was slightly depressed at 1000 mg/kg bw/day between postnatal days 0 and 4. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/female rats: 300 mg/kg bw/day
NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 300 mg/kg bw/day

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 Sika Hardener MTJ 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 doses 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. Sika Hardener MTJ concentrations in the dosing formulations varied in the acceptable range between 98 % and 108 % 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 43 days). Females were additionally exposed through the gestation period and up to lactation days 3 -6, i.e. up to the day before necropsy (altogether for 43-58 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. 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 just after found dead or 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. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups. The kidneys of male and female animals in the mid dose group selected for general toxicological examinations were processed and evaluated histopathologically due to the renal changes observed in animals of the high dose.

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.

Mortality

Three male animals, three pregnant females during gestation and one dam during lactation, which were administered with 1000 mg/kg bw/day, were found dead during the course of the study. There were no preceding clinical signs in any of these animals. Histopathological examinations revealed hyaline droplet nephropathy and tubular necrosis in the kidneys, congestion and alveolar edema in the lungs as cause of death in all of these animals. These lesions were considered as related to the application of the high dose of the test item.

Clinical observation

Adverse clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations. The behavior and physical condition of the animals was not impaired at each dose level (1000, 300 or 100 mg/kg bw/day) during the entire treatment period.

Body weight and body weight gain

The body weight development was slightly reduced at 1000 mg/kg bw/day with respect to their controls in male animals considering the whole administration period and in female animals during the lactation period.

Food consumption

The daily mean food consumption was slightly reduced in male and female animals at 1000 mg/kg bw/day during week 1 and in female animals at 1000 mg/kg bw/day during the course of gestation and lactation periods.

Hematology

No test item-related changes were observed in investigated hematology or blood coagulation parameters.

Clinical chemistry

Clinical chemistry examinations did not reveal any adverse changes in the examined parameters.

Necropsy

Specific macroscopic alterations related to the test item were not found at necropsy.

Organ weight

There were no test item related changes in brain, testes and epididymides weights of male animals at any dose level. There were slight increases in the absolute and/or relative weights of the kidneys at 1000 mg/kg bw/day (males and females randomly selected) However, the values were within the historical control ranges.

Histopathology

In the kidneys, hyaline droplet nephropathy and tubular necrosis, in the lungs, congestion and alveolar edema were detected in all prematurely died animals at 1000 mg/kg bw/day.

Hyaline droplet nephropathy was also observed in the kidneys of all examined male animals at 1000 mg/kg bw/day randomly selected for examination. Renal alterations in the form of hyaline droplet nephropathy or tubular necrosis were not detected in the female animals at 1000 mg/kg bw/day or in the male and female animals belonging to the 300 mg/kg bw/day group.

Histopathological examinations did not reveal any test item related changes in male or female genital organs (ovaries, testes and epididymides) or in pituitary in at 1000 mg/kg bw/day.

Reproduction

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.

Offspring

The offspring’s body weight development was slightly depressed between postnatal days 0 and 4. There was no increased mortality and no adverse clinical or necropsy findings were detected in the offspring terminated as scheduled.

Conclusion

Under the conditions of the present study, Sika Hardener MTJ administered orally (by gavage) at 1000 mg/kg bw/day caused premature death and hyaline droplet nephropathy and tubular necrosis in the kidneys in male (3/12) and female (4/12) Hsd.Brl.Han: Wistar rats.

Slightly reduced body weight development, food consumption, increases in the kidneys weights (males and females) and hyaline droplet nephropathy (males) were observed in surviving animals at 1000 mg/kg bw/day. There was no adverse influence on the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats at 1000 mg/kg bw/day.

At 300 and 100 mg/kg bw/day, there were no test item related adverse alterations or impairment of the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female animals.

The offspring’s body weight development was slightly depressed at 1000 mg/kg bw/day between postnatal days 0 and 4.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 300 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 300 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline and GLP 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

The purpose of a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was to obtain initial information on the toxic potential of Sika Hardener MTJ 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 doses 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. Sika Hardener MTJ concentrations in the dosing formulations varied in the acceptable range between 98 % and 108 % 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 43 days). Females were additionally exposed through the gestation period and up to lactation days 3 -6, i.e. up to the day before necropsy (altogether for 43-58 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. 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 just after found dead or 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. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups. The kidneys of male and female animals in the mid dose group selected for general toxicological examinations were processed and evaluated histopathologically due to the renal changes observed in animals of the high dose.

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.

Mortality

Three male animals, three pregnant females during gestation and one dam during lactation, which were administered with 1000 mg/kg bw/day, were found dead during the course of the study. There were no preceding clinical signs in any of these animals. Histopathological examinations revealed hyaline droplet nephropathy and tubular necrosis in the kidneys, congestion and alveolar edema in the lungs as cause of death in all of these animals. These lesions were considered as related to the application of the high dose of the test item.

Clinical observation

Adverse clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations. The behavior and physical condition of the animals was not impaired at each dose level (1000, 300 or 100 mg/kg bw/day) during the entire treatment period.

Body weight and body weight gain

The body weight development was slightly reduced at 1000 mg/kg bw/day with respect to their controls in male animals considering the whole administration period and in female animals during the lactation period.

Food consumption

The daily mean food consumption was slightly reduced in male and female animals at 1000 mg/kg bw/day during week 1 and in female animals at 1000 mg/kg bw/day during the course of gestation and lactation periods.

Hematology

No test item-related changes were observed in investigated hematology or blood coagulation parameters.

Clinical chemistry

Clinical chemistry examinations did not reveal any adverse changes in the examined parameters.

Necropsy

Specific macroscopic alterations related to the test item were not found at necropsy.

Organ weight

There were no test item related changes in brain, testes and epididymides weights of male animals at any dose level. There were slight increases in the absolute and/or relative weights of the kidneys at 1000 mg/kg bw/day (males and females randomly selected) However, the values were within the historical control ranges.

Histopathology

In the kidneys, hyaline droplet nephropathy and tubular necrosis, in the lungs, congestion and alveolar edema were detected in all prematurely died animals at 1000 mg/kg bw/day.

Hyaline droplet nephropathy was also observed in the kidneys of all examined male animals at 1000 mg/kg bw/day randomly selected for examination. Renal alterations in the form of hyaline droplet nephropathy or tubular necrosis were not detected in the female animals at 1000 mg/kg bw/day or in the male and female animals belonging to the 300 mg/kg bw/day group.

Histopathological examinations did not reveal any test item related changes in male or female genital organs (ovaries, testes and epididymides) or in pituitary in at 1000 mg/kg bw/day.

Reproduction

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.

Offspring

The offspring’s body weight development was slightly depressed between postnatal days 0 and 4. There was no increased mortality and no adverse clinical or necropsy findings were detected in the offspring terminated as scheduled.

Conclusion

Under the conditions of the present study, Sika Hardener MTJ administered orally (by gavage) at 1000 mg/kg bw/day caused premature death and hyaline droplet nephropathy and tubular necrosis in the kidneys in male (3/12) and female (4/12) Hsd.Brl.Han: Wistar rats.

Slightly reduced body weight development, food consumption, increases in the kidneys weights (males and females) and hyaline droplet nephropathy (males) were observed in surviving animals at 1000 mg/kg bw/day. There was no adverse influence on the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats at 1000 mg/kg bw/day.

At 300 and 100 mg/kg bw/day, there were no test item related adverse alterations or impairment of the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female animals.

The offspring’s body weight development was slightly depressed at 1000 mg/kg bw/day between postnatal days 0 and 4.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 300 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 300 mg/kg bw/day

Short description of key information:
Key study: The purpose of a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was to obtain initial information on the toxic potential of Sika Hardener MTJ and its possible effects on reproductive performance. The test item administered orally (by gavage) at 1000 mg/kg bw/day caused premature death and hyaline droplet nephropathy and tubular necrosis in the kidneys in male (3/12) and female (4/12) Hsd.Brl.Han: Wistar rats. There was no adverse influence on the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats at 1000 mg/kg bw/day. Also at 300 and 100 mg/kg bw/day, there were no test item related adverse alterations or impairment of the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female animals.
The offspring’s body weight development was slightly depressed at 1000 mg/kg bw/day between postnatal days 0 and 4.
Based on the observations of the study the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/female rats: 300 mg/kg bw/day
NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 300 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:
Guideline and GLP study

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.As a result the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.