Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethyl]-.omega.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethoxy]-

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The reprotoxic properties of the test item were assessed in a study performed according to OECD Guideline 421 in rats. Based on the results obtained from testing the parental NOAEL, the NOAEL for reproductive performance and the NOAEL for the F1 generation were determined to be 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:

2013-02-14 to 2013-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test, EPA Health Effects Test Guidelines, July 2000.

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Hsd.Brl.Han:Wist

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: 79- 89 days old, Female animals: 77 - 84 days old
Satellite animals
Male animals: 83- 87 days old
Female animals: 83 - 87 days old
- Weight at study initiation:
Male animals: 297 - 371 g
Female animals: 187 - 239 g
- Housing: Type II polypropylene/polycarbonate
Size: 22 x 32 x 19 cm (width x length x height)
Supplier: Charles River Europe
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: individually
- Diet (e.g. ad libitum):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 (e.g. ad libitum):Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:14 days, for satellite animals: 40 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on exposure:

PREPARATION 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 beforehand not longer than for three days.


VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item is not soluble in water; therefore sunflower oil was used as a vehicle
- Amount of vehicle (if gavage):
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.

Details on mating procedure:

Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females were cohabited with the same male until copulation occurred. A second mating period was implemented due to insufficient number of litters in the 100 mg/kg bw/day group after the fist mating period. Additional 12 animals /sex were involved in the control and 100 mg/kg bw/day groups in the study to ensure the appropriate number of litters determined in the test guideline OECD 421.
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 421). Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Concentration of the test item in the dosing formulations was checked twice during the study. Sika Hardener LJ concentrations in the dosing formulations varied in the range of 91 and 103 % of the nominal values at all analytical occasions, thereby confirming proper dosing.

Duration of treatment / exposure:

The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology except one not delivered female in group 100 mg/kg bw/day, which was administered on the day of the necropsy by a mistake. Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks.
Male animals were dosed for 54 days in the main study and for 42 days in the satellite groups and then they were subjected to necropsy one day after the last treatment. Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 2 - 6 (altogether for 41 – 60 days in the main study and for 42 – 46 days in the satellite groups, depending on day of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Dead female animal was treated altogether for 27 days.
Non-pregnant and not delivered female animals were treated up to and including the day before necropsy (altogether for 41 or 43 days). Females with full intrauterine death (not delivered) were subjected to necropsy on the gestation day 24. Control animals were handled in an identical manner to the test groups receiving only 5 mL vehicle/kg bw. F1 offspring was euthanized on postnatal day 4.

Frequency of treatment:

Animals were treated once per day.

Details on study schedule:

Male animals were dosed for 54 days in the main study and for 42 days in the satellite groups and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 2 - 6 (altogether for 41 – 60 days in the main study and for 42 – 46 days in the satellite groups, depending on day of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Dead female animal was treated altogether for 27 days.
Non-pregnant and not delivered female animals were treated up to and including the day before necropsy (altogether for 41 or 43 days). Females with full intrauterine death (not delivered) were subjected to necropsy on the gestation day 24.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals/sex in the control and dose groups

Control animals:

yes, concurrent vehicle

Details on study design:

The dose setting is based on findings obtained in a previous 28-Day oral gavage dose range finding study with Urea 1 in rats (results were provided by the Sponsor) and in agreement with the Sponsor. Doses are 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:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made at the times of weekly weighing, prior to and during the mating until necropsy. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.

BODY WEIGHT: Yes
All parent animals were weighed with an accuracy of 1 g. 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 20 and on postpartal days 0 (within 24 hours after parturition) and 4, as well as on the day of necropsy. Body weight of the female animals was weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

Oestrous cyclicity (parental animals):

Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary.
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 421). Sperm positive females were caged individually.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and weighed within 24 hours of parturition (on the day when parturition was complete) and day 4 post-partum with an accuracy of 0.01 g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all pups found dead to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.

Postmortem examinations (parental animals):

Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isoflurane and then were exsanguinated. 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. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides, prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

Postmortem examinations (offspring):

GROSS NECROPSY
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4
Mean body weight gain per litter between postnatal days 0-4
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
Survival Index of pups on postnatal day 4
Sex ratio % (on postnatal days 0 and 4)

Statistics:

The statistical evaluation of appropriate data 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.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”.

Reproductive indices:

The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"

Offspring viability indices:

The offspring viability indices were calculated: survivla index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Daily Observations
Test item related salivation was observed in male animals of 1000, 300 and 100 mg/kg bw/day. The behavior and physical condition of animals in 1000, 300, and 100 mg/kg bw/day and in the control group were considered to be normal during the entire observation period.
Salivation appeared in male animals in a dose related manner (12/12, 6/12 and 2/24, respectively to doses) regarding the onset, duration and severity. One control male animal (1/24) and one female of 100 mg/kg bw/day (1/24) also showed salivation, which was related to the treatment but independent from the test item.
Piloerection, vaginal bleeding, pale skin and cold body temperature were noted for one dam of control group (1/22) during the parturition as signs of elaborated delivery.
Alopecia and scab were observed on one dam of 100 mg/kg bw/day (1/19) from gestation day 21 and lactation day 1, respectively. Alopecia and scab are common findings in this strain of experimental rats and is seen also in untreated rats. Therefore, this isolated finding was considered to be independent from treatment.

Detailed Weekly Observations
The behavior and physical condition of animals was not affected by the test item at any dose level (1000, 300 or 100 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.
Alopecia and scab as described above were detected in female animal at the detailed weekly observation, too.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no test item related mortality in any group during the course of study.
One control female animal was found dead on gestation day 10 (study Day 27). There were no preceding clinical signs. Necropsy and histopathological examination revealed individual lesions (greyish white substance on the pleura and in the thoracic cavity; subacute serous-fibrinous pleuritis and epicarditis) as the cause of death. These findings may refer to a para-gastric treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight gain was slightly reduced in male and female animals of 1000 mg/kg bw/day. However this slight change did not resulted in significant changes in the mean body weight of this group.
More specifically, compared to the control group, statistical significances were detected at the slightly less body weight gain of male animals of 1000 mg/kg bw/day group on weeks 2, 4 and 8 resulting in a slightly less summarized mean body weight gain.
Statistical significances were also noted with respect to the control for the less mean body weight gain of female animals in 1000 mg/kg bw/day group between Days 7 and 13 and between Days 0 and 14. However these slight differences did not cause significant differences in the mean body weight values in male or female animals with respect to the appropriate control, therefore were not considered to be of toxicologically relevance.
Sporadic statistically significances were also detected in male animals of 300 mg/kg bw/day group for a less body weight gain on week 4 and 8 and for a higher body weight gain on week 5. The body weight gain exceeded the control value in female animals of 300 mg/kg bw/day group on gestation week 1 and in 100 mg/kg bw/day during the premating period (between days 0 and 13). The body weight was slightly less in dams of 1000 mg/kg bw/day group, and it was higher with respect to the control in 300 mg/kg bw/day group on lactation day 0. Although being statistically different to the concurrent control group, these transient differences in the body weight gain were judged to be indicative of biological variation and not related to the test item in the lack of any dose relevance or due to the small degree.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

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).
Compared to the control animals, statistical significances were observed in the slightly reduced mean food consumption of male animals in 100 mg/kg bw/day on week 2, in the higher mean food consumption of female animals of 300 mg/kg bw/day group during the gestation weeks 1 and 2 and in of 100 mg/kg bw/day groups during the pre-mating period (weeks 1 and 2). However these slight but statistically significant changes were not related to doses, were with small degree and similar findings were not detected for the animals of the high dose group. Therefore these were considered to be without any toxicological relevance.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Histological examination of male and female genital organs (testes, epididymides and ovaries) did not reveal any toxic or other test item related alterations at the highest dose of 1000 mg/kg/bw/day.

In dead female control animal (1/24), subacute serous-fibrinous pleuritis and epicarditis were observed as the cause of death. These findings were considered to be individual disease. No degenerative or other lesions were observed in the other investigated organs.
The investigated organs of reproductive system (testes, epididymides) were histologically normal in all animals of the 1000 mg/kg bw/day dose (12/12) and in all male animals which did not fertilize females in 100 mg/kg bw/day group (4/4). 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 for all dose groups. Compared to the concurrent control, epididymides were histologically normal in all animals.
The ovaries had a normal structure characteristic for the species, age and phase of sexual cycle in all examined female animals of 1000 mg/kg bw/day group and non-pregnant animals of 100 mg/kg bw/day group. 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 were normal in all cases as well. The ovarian cyst was an individual alteration in one female animal of the 1000 mg/kg bw/day (1/10 dam), which is a common findings in experimental rats.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

The delivery data of dams were not affected by the treatment with the test item at 1000, 300 or 100 mg/kg bw/day doses.
The mean number of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss and total intrauterine mortality were similar in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the mean duration of pregnancy, in the mean number of total births, live-borns, stillborns or viable pups.
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 doses in male or female animals.
Compared to the concurrent control groups, statistical significance was noted for the higher fertility index of male and female animals of 300 mg/kg bw/day group, which included also a higher percentage of fertile animals and a less percentage of infertile animals.
Statistical significances were noted for the higher percentage of pregnant females and for the less percentage of non-pregnant females in the 300 mg/kg bw/day groups comparing to the control group.
These slight, but statistically significant differences in the fertility indices, in the percentage of pregnant and non-pregnant females, and gestation index with respect to the control were considered to be indicative of biological variation and not related to the test item treatment.

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 observed

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring.
The number and percentage of pups with findings (cold, no or little milk in the stomach) were similar in the control and test item treated groups. Hemorrhage was observed on the left hind limb of one offspring (1/96) in 1000 mg/kg bw/day group. Hemorrhage occurred with low incidence and it can be observed in not treated offspring therefore was not considered to be of toxicologically relevance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on pup’s mortality.
The mean number of live births per litter, the mean number of viable pups per litter on postnatal days 0 and 4 were similar in all groups. There were no differences between the control and test item treated (1000, 300 or 100 mg/kg bw/day) groups in the survival indices.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Test item effect on the body weight development of offspring was not found.
The mean litter and offspring weights, the mean litter and offspring weight gains were similar in the control and in all test item treated groups on postnatal days 0 and 4.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Description (incidence and severity):

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.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related macroscopic alterations were found in offspring subjected to gross pathological examination.

Histopathological findings:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Reproductive effects observed:

no

Conclusions:

The reprotoxic properties of the test item were assessed in a study performed according to OECD Guideline 421 in rats. Based on the results obtained from testing the parental NOAEL, the NOAEL for reproductive performance and the NOAEL for the F1 generation were determined to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this reproduction/developmental toxicity screening test was to provide initial information concerning the effect of the test item Sika Hardener LJ on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 1000, 300 and 100 mg/kg bw/day at concentrations of 200 mg/mL, 60 and 20 mg/mL corresponding to 5 mL/kg bw dose volume. Groups of satellite animals (12 animals/sex/group; control and 100 mg/kg bw/day group) were involved in the study to ensure the appropriate number of litters at the low dose. Control animals were administered with 5 mL/kg bw volume sunflower oil. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Concentration of the test item in the dosing formulations was checked twice during the study. Sika Hardener LJ concentrations in the dosing formulations varied in the range of 91 and 103 % of the nominal values at all analytical occasions, thereby confirming proper dosing. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy (altogether for 54 days in the main groups and for 42 days in the satellite groups). For females, test item was administered through the gestation period and up to lactation days 2-6 i.e. up to the day before the necropsy (altogether for 41 - 60 days).

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. The dams were allowed to litter, and rear their young up to day 4 postpartum. 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 and ovaries) in the control and high dose groups. The reproductive organs of non-pregnant females and males cohabited with in the low and mid dose groups were also processed and evaluated histologically. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with the vehicle only.

Results

Mortality

There was no test item related mortality at any dose level (1000, 300 and 100 mg/kg bw/day). One control female animal was found dead due to individual disorder on Day 27.

Clinical observation

Toxic signs related to the test item were not detected at any dose level at the daily and detailed weekly clinical observations. Salivation was observed in the male animals of the 1000, 300 and 100 mg/kg bw/day groups with variable frequency within a group but in a dose related manner regarding the incidence, onset and degree. The behavior and physical condition of animals were normal during the entire observation period (pre-mating and post mating for male animals, during pre-mating for dams and non-pregnant females, during gestation and lactation periods for dams).

Body weight and body weight gain

The mean body weight gain of male animals in 1000 mg/kg bw/day group was slightly reduced during the observation period. However this slight change did not resulted in significant changes in the mean body weight of this group therefore was not judged to be toxicologically relevant.

Food consumption

The mean daily food consumption was not affected by the test item in male or female animals at 1000, 300 and 100 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods).

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.

Necropsy

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

Organ weight

There were no test item related changes in brain, testes and epididymides weights of male animals at any dose level.

Histopathology

Histopathological examinations of male and female genital organs (ovaries, testes and epididymides) did not reveal any toxic or other test item related changes at any dose level.

Offspring

Negative effects of the test item on offspring development (mortality, clinical signs, and body weight and necropsy findings) were not detected between postnatal days 0 and 4.

Conclusion

Under the conditions of the present study Sika Hardener LJ did not cause toxic changes and did not influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 post-partum after repeated dose oral administration at 1000, 300 or 100 mg/kg bw/day.

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

NOAEL for male rats: 1000 mg/kg bw/day

NOAEL for female rats:1000 mg/kg bw/day

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

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

NOAEL for F1 Offspring:1000 mg/kg bw/day

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:

GLP and guideline compliant study.

Additional information

The purpose of this reproduction/developmental toxicity screening test was to provide initial information concerning the effect of the test item Sika Hardener LJ on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 1000, 300 and 100 mg/kg bw/day at concentrations of 200 mg/mL, 60 and 20 mg/mL corresponding to 5 mL/kg bw dose volume. Groups of satellite animals (12 animals/sex/group; control and 100 mg/kg bw/day group) were involved in the study to ensure the appropriate number of litters at the low dose. Control animals were administered with 5 mL/kg bw volume sunflower oil. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Concentration of the test item in the dosing formulations was checked twice during the study. Sika Hardener LJ concentrations in the dosing formulations varied in the range of 91 and 103 % of the nominal values at all analytical occasions, thereby confirming proper dosing. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy (altogether for 54 days in the main groups and for 42 days in the satellite groups). For females, test item was administered through the gestation period and up to lactation days 2-6 i.e. up to the day before the necropsy (altogether for 41 - 60 days).

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. The dams were allowed to litter, and rear their young up to day 4 postpartum. 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 and ovaries) in the control and high dose groups. The reproductive organs of non-pregnant females and males cohabited with in the low and mid dose groups were also processed and evaluated histologically. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with the vehicle only.

Mortality

There was no test item related mortality at any dose level (1000, 300 and 100 mg/kg bw/day). One control female animal was found dead due to individual disorder on Day 27.

Clinical observation

Toxic signs related to the test item were not detected at any dose level at the daily and detailed weekly clinical observations. Salivation was observed in the male animals of the 1000, 300 and 100 mg/kg bw/day groups with variable frequency within a group but in a dose related manner regarding the incidence, onset and degree. The behavior and physical condition of animals were normal during the entire observation period (pre-mating and post mating for male animals, during pre-mating for dams and non-pregnant females, during gestation and lactation periods for dams).

Body weight and body weight gain

The mean body weight gain of male animals in 1000 mg/kg bw/day group was slightly reduced during the observation period. However this slight change did not resulted in significant changes in the mean body weight of this group therefore was not judged to be toxicologically relevant.

Food consumption

The mean daily food consumption was not affected by the test item in male or female animals at 1000, 300 and 100 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods).

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.

Necropsy

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

Organ weight

There were no test item related changes in brain, testes and epididymides weights of male animals at any dose level.

Histopathology

Histopathological examinations of male and female genital organs (ovaries, testes and epididymides) did not reveal any toxic or other test item related changes at any dose level.

Offspring

Negative effects of the test item on offspring development (mortality, clinical signs, and body weight and necropsy findings) were not detected between postnatal days 0 and 4.

Conclusion

Under the conditions of the present study Sika Hardener LJ did not cause toxic changes and did not influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 post-partum after repeated dose oral administration at 1000, 300 or 100 mg/kg bw/day.

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

NOAEL for male rats: 1000 mg/kg bw/day

NOAEL for female rats:1000 mg/kg bw/day

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

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

NOAEL for F1 Offspring:1000 mg/kg bw/day

Justification for classification or non-classification

Based on results obtained in reproduction toxicity testing, the test item was not classified or labeled according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.