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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 March 2018 - 8 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 March 2018 - 8 May 2018
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:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity w ith the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
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
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Male animals: 11-12 weeks (80-90 days) old
Female animals: 11-12 weeks (80-90 days) old
- Weight at study initiation:
Male animals: 349 – 397 g
Female animals: 187 – 235 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate cages
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females housed individually.
- Diet: ad libitum, 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
- Water: ad libitum, tap water
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 6 mg/mL, 20 mg/mL and 60 mg/mL. Each formulation were prepared by careful weight measurement just prior to administration. Formulations were prepared daily and administered within a short period thereafter thus the stability of the formulations is considered to be not relevantly affected.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore PEG 400 will be used for preparing formulations appropriate for oral administration. PEG 400 is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 6 mg/mL, 20 mg/mL and 60 mg/mL
- Treatment volume: 5 mL/kg bw
- Lot/batch no.: 16I284004
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurs or 14 days have elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility
- After successful mating each pregnant female will be caged individually.
Analytical verification of doses or concentrations:
yes
Remarks:
ICP-OES
Details on analytical verification of doses or concentrations:
Five samples were taken (from different places) from each concentration (Groups 2, 3 and 4) and at least three parallels are measured twice during the study. Similarly, five samples were taken from the control solution (Group 1). Due to the solubility and instability feature of the test item, stability determination was not possible.
Duration of treatment / exposure:
Males dosed for at least 28 days (14 days pre-mating and 14 days mating plus an optional extended post-mating period until the necessary number of pregnant female animals is evident)
Females dosed for 14 days pre-mating, through 14 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose setting is based on findings obtained in a 14-day dose range finding study performed with the substance. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
Observations 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 behavior pattern, changes in gait, posture and response to handling. Special attention will be directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted on five male and five female animals randomly selected from each group during their respective last exposure week but before the blood sampling.

BODY WEIGHT: Yes
Parental males weighed on the first day of dosing (day 0), weekly thereafter and at termination. Parental females weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals additionally measured on gestation day 10 in order to give accurate treatment volumes, but these data was evaluated statistically. Body weight data was reported individually for adult animals. Body weight was measured on the day of necropsy for animals subjected to organ weighing (all male animals and females selected for further examinations).

FOOD CONSUMPTION: Yes
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase as follows: premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals. Fasted body weight was determined for animals selected for toxicity examinations before the necropsy.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks. Animals exhibiting typical 4-5 days cycles were included in the study preferably. Vaginal smears were prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.
Vaginal smear were prepared on the day of the necropsy.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- testis weight, epididymis weight, prostate weight and seminal vesicles with coagulating glands as a whole
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) presence of gross anomalies. Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete, post-natal day 0) and on postnatal day 13. The anogenital distance of each pup was determined on postnatal day 4. Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum T4 and TSH levels. The number of nipples/areolae in male pups was counted on postnatal day 13.

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
CLINICAL PATHOLOGY
Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) 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 hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry.
In addition, blood samples were collected for determination of serum levels of thyroid hormones (T4 and TSH).

HEMATOLOGY
WBC (White Blood Cell leukocyte count), RBC (Red Blood Cell erythrocyte count), HGB (Hemoglobin concentration), HCT (Hematocrit, relative volume of erythrocytes), MCV (Mean Corpuscular erythrocyte Volume), MCH (Mean Corpuscular erythrocyte Hemoglobin), MCHC (Mean Corpuscular erythrocyte Hemoglobin Concentration), PLT (Platelet thrombocyte count), RET (Reticulocytes), Differential white blood cell count

CLINICAL CHEMISTRY
ALT (Alanine Aminotransferase activity), AST (Aspartate Aminotransferase activity), TBIL (Total Bilirubin concentration), CREA (Creatinine concentration),
UREA (Urea concentration), GLUC (Glucose concentration),CHOL (Cholesterol concentration), Na+ (Sodium concentration),K+ (Potassium concentration), ALB (Albumin concentration), TPROT (Total Protein concentration)

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (T4,TSH) as follows:
- from all dams and at least two pups per litter on day 13 if feasible
- from all parent male animals at termination

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The appearance of the tissues and organs were observed, and any abnormality was recorded including details of the location, color, shape and size. At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals was determined. In addition, for five males and females randomly selected from each group, adrenal glands,brain, heart, kidneys, liver, spleen and thymus was weighed. Absolute organ weight was reported. Relative organ weight (to body and brain weights) was calculated and reported.
The thyroid weight was determined after fixation.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments ofthe ovary as well as the epithelial capsule and ovarian stroma.
The following organs were preserved: Adrenal glands, Aorta, Bone with marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Eyes (lachrymal gland with Harderian glands), Female mammary gland, Gonads (testes with epididymides, ovaries, uterus with vagina), Gross lesions, Heart, Kidneys, Large intestines (caecum, colon, rectum, including Peyer’s patches), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes (submandibular, mesenteric), Muscle (quadriceps), Esophagus, Pancreas, Pituitary, Prostate, Salivary glands (submandibular), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, Small intestines(representative regions: duodenum, ileum, jejunum), Spinal cord (at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder.

At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals was determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus was weighed. Absolute organ weight was reported. Relative organ weight (to body and brain weights) was calculated and reported. The thyroid weight was determined after fixation.

Histopathological examinations were performed for the pancreas in all dose groups.





Postmortem examinations (offspring):
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test). Dead pups found were subjected to necropsy by a macroscopic examination. Any observed abnormalities were recorded.
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.
Reproductive indices:
see table 1
Offspring viability indices:
see table 2
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Soft stool caused by the vehicle was detected in the bedding material in each cage: male and female animals of control, 30, 100 and 300 mg/kg bw/day groups from Day 2 up to the termination of the treatment. Protrusion (~6 mm) at the vagina connected with a tissue formation was observed for one female animal at 300 mg/kg bw/day between lactation days 0 and 15. This clinical sign was considered to be individual finding and not related to the treatment with the test item.
There were no clinical signs in the other animals (male or female) of control, 30, 100 or 300 mg/kg bw/day groups, i.e. these animals exhibited normal behavior and physical condition with no abnormalities during the treatment period.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the control, 30, 100 or 300 mg/kg bw/day groups (male or female) during the course of study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight development was not adversely affected by the test item in male or female animals at 30, 100 or 300 mg/kg bw/day during the entire treatment period. There were no significant differences between the control and the test item treated male animals in the mean body weight although the mean body weight gain was significantly lower than in the control animals at 100 mg/kg bw/day between Days 13 and 20, and it was significantly higher than in the control group at 30 and 300 mg/kg bw/day between Days 41 and 48.
There were no statistically significant differences between the control and test item treated groups in the mean body weight and mean body weight gain in female animals at 30, 100 and 300 mg/kg bw/day in the premating, gestation or lactation periods.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item or treatment related adverse changes in the food consumption of male and female animals at any dose level (30, 100 and 300 mg/kg bw/day).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, statistical significances were detected at the slightly lower mean red blood cell (erythrocyte) count (RBC) and mean hematocrit value at 300 mg/kg bw/day. In female animals, slightly but statistically significantly lower mean red blood cell (erythrocyte) count (RBC) was observed at 300 mg/kg bw/day. The statistically significant differences between the control and high dose treated groups for red blood cell parameters (RBC, HCT) were considered to have no toxicological relevance due to the minor degree and lack of any changes in other parameters. Furthermore, the values are still well within the historical control ranges, respectively.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, statistical significance was detected with respect to their control at the lower mean total bilirubin (TBIL) and mean creatinine concentration (CREA) at 300 mg/kg bw/day. These differences between control and test item treated male animals reached statistical significances however individual values were in line with the historical control and these changes were judged to have no toxicological relevance. All examined clinical chemistry parameters were comparable in female animals in the control and in all test item treated groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observations did not demonstrate any test item related adverse changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (30, 100 and 300 mg/kg bw/day, control).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examinations did not reveal any test item related adverse alterations in the reproductive organs or tissues of male or female animals at 300 mg/kg/bw/day (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) Histological examination of the pancreas revealed increased amount of zymogen granules and flattened and basophilic acinar cells at 100 mg/kg bw/day (1/6 male) and at 300 mg/kg bw/day (5/5 male and 3/5 female) in mild or moderate degree. These findings were in accordance with the macroscopic changes in the pancreas (enlargement and paleness). No histopathological findings were reported in the pancreas in the low dose group (30 mg/kg bw/day). The increased amount of zymogen granules and the flattened and basophilic acinar cells – without acinar cell necrosis, atrophy or disorganization of acinar structure – was considered as histological signs of an adaptive response of the pancreatic acinar cells to test item treatment indicative of an adaptive acinar hypertrophy.
These findings could be caused by an enhanced synthesis and secretion of proenzymes induced by the test item. The proenzymes are inactive precursors which do not induce adverse effects in acinar cells after prolonged storage. So, the correlation between the gross pathological findings and histological picture is obvious, but the toxicological significance is questionable. Since neither inflammation, necrosis or apoptosis were observed nor an increase in severity over time (refer to 14-day Dose Range Finding Study; Study no. 666-400-0899), reversibility of these exocrine pancreatic observations can be considered as very likely. Overall, these observations are likely to be interpreted adaptive changes induced by the test item and no adversity is to be expected. The endocrine pancreas was not affected and cytomorphology of Langerhans-islets was normal in all cases. In conclusion, all observations in the pancreas are interpreted as non-adverse.
In all male animals (12/12 control, 12/12 at 300 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism in all cases.
Decreased amount of secrete in the seminal vesicle (one side) was observed in two male animals (2/2) at 100 mg/kg bw/day in accordance with macroscopic findings (smaller than normal). This finding was considered as individual disorder without toxicological relevance as there were no accompanying inflammatory or degenerative lesions. In the female animals (12/12 control, 11/12 at 300 mg/kg bw/day) the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. The cortical region of ovaries 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. In one female (1/1) at 300 mg/kg bw/day metritis was observed in the uterus. This finding was considered as individual disease, not related to the test item treatment. In one female animal (1/1) at 100 mg/kg bw/day dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
Alveolar emphysema in minimal or mild degree was observed in the lungs in the control (1/5 male and 2/5 dams) and at 300 mg/kg bw/day (1/5 male). Hyperplasia of bronchus associated lymphoid tissue (BALT) was noted for single animals in the control group (1/5 male, 1/5 dam). Acute hemorrhage was observed in the thymus in one male at 300 mg/kg bw/day (1/1). Severe hydronephrosis (one side) was observed in one male animal at 100 mg/kg bw/day (1/1). Alveolar emphysema in the lungs was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Hyperplasia of bronchus associated lymphoid tissue (BALT) is a physiological immune-morphological phenomenon, without toxicological significance. Thymic hemorrhage was a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the stomach, the small and large intestines, the liver, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the selected animals. The cyto-morphology of the endocrine glands was the same in the control and the treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Serum Levels of Thyroid Hormones
There were no significant differences with respect to the control in the TSH thyroid hormone levels in parental male animals or in offspring sampled on postnatal day 13 at any dose levels. Statistically significant difference with respect to the control was noted for the slightly higher mean thyroid hormone (free T4) level in PN13 offspring at 100 and 300 mg/kg bw/day. This minor difference was considered to be toxicologically not relevant as the individual values were within the historical control range (mean: 2.46±0.35 ng/dL; n=87; min: 1.77 ng/dL; max: 3.73 ng/dL). Moreover, since TSH levels revealed no differences when compared to the control and there were no findings in regards to histopathological examinations of respective organs, there is no indication that hypothalamic–pituitary–thyroid (HPT) axis was affected by the test item.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrous cycle was not affected by the test item at any dose level (30, 100 or 300 mg/kg bw/day). There were no significant differences between the control and treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus during the pre-experimental or pre-mating period.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no significant differences between the control and test item treated (30, 100 and 300 mg/kg bw/day) groups in the copulatory and fertility indices (males and females) and in the gestation indices (females). The percentage of pregnant females, dams delivered, pregnants with liveborn(s) and the pre-coital interval and the mean number of conceiving days were comparable in female animals of control and test item treated animals.
Delivery Data of Dams
There were no relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (30, 100 or 300 mg/kg bw/day). The mean number of implantation sites per dams and the mean of postimplantation loss were comparable 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 on post-partal day 0, in the percentage of dams with viable offspring on postpartal day 0 or in the live birth index.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse clinical signs in the offspring between post-natal days 0 and 13. The percentage of offspring with signs (cold, not suckled) at 30, 100 and 300 mg/kg bw/day was higher than in the control group on post-natal day 0. However, these findings were not considered to be toxicologically relevant as no dose relevance was observed and the signs were transient – detected shortly after the delivery. Moreover, these observations were not associated with any influence on the development of the offspring. Occasionally, smaller than normal pup (1% at 30 and 100 mg/kg bw/day) was also observed which were considered to have no toxicological relevance.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality. The mean number of dead offspring (including missing pups) per litter was comparable in the control and test item treated groups on post-natal day 0 and between post-natal day 0 and 13. The survival indices were similar in the control and test item treated (30, 100 or 300 mg/kg bw/day) groups. The mean number of live pups per litter and the mean number of viable pups per litter were comparable in all groups on postnatal days 0, 4 and 13.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A test item related effect on the body weight development of the offspring was not detected. There were no statistical significances between the control and test item treated (30, 100 and 300 mg/kg bw/day) groups in the mean litter weight and litter weight gain on post-natal days 0, 4 and 13. Statistical significance was observed in the mean pup weight on post-natal day 4 and in the mean pup weight gain between post-natal days 0 and 4 at 300 mg/kg bw/day. Considering the offspring’s body weight in males and females, separately, statistically significant difference with respect to the control was detected for the lower male and female pup weight at 100 and 300 mg/kg bw/day on post-natal day 4. The differences in the pup’s weight were of minor degree and therefore considered to have no or little toxicological relevance. Moreover, the mean litter weights were unremarkable in all treated groups. Furthermore, the reduced finding was transient, as the mean pup body weight on PND 13 did not reveal any differences to the control any more. This could be due to litter standardization on PND 4.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances in male or female offspring were not affected by the test item at 30, 100 and 300 mg/kg bw/day. The anogenital distances, both absolute and normalized, were comparable in the control and test item treated groups in male and female pups.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The nipple retention in male offspring was not affected by the test item at 30, 100 and 300 mg/kg bw/day.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination. There were no macroscopic changes in the organs or tissues of dead pups (3/3 in the control) on post-natal day 0. There were no macroscopic changes in the organs or tissues of stillborn offspring (2/2 at 30 mg/kg bw/day, and 1/1 at 300 mg/kg bw/day) subjected to necropsy on post-natal day 0. One of them in the low dose was uncleaned and its umbilical cord was intact.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex Distribution
There were no test item related differences between the control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.
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: highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Under the conditions of the present study, Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyl- trimethoxysilane did not adversely influence the fertility or reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 30, 100 and 300 mg/kg bw/day doses administered by oral gavage. The NOAEL for systemic toxicity of male/female rats was 300 mg/kg bw/day, the NOAEL for reproductive performance of male/female rats was 300 mg/kg bw/day and the NOAEL for F1 Offspring was 300 mg/kg bw/day, the highest dose tested.
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422 was performed to obtain initial information on the toxic potential of Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane 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 13 post- partum when repeatedly administered orally (by gavage) to parental animals at doses of 30 mg/kg bw/day, 100 mg/kg bw/day and 300 mg/kg bw/day compared to control animals. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 30, 100 and 300 mg/kg bw/day doses corresponding to concentrations of 0, 6, 20 and 60 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. Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane concentrations in the dosing formulations varied in the acceptable range between 101 % and 109 % 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 50 days). Females were additionally exposed through the gestation period and up to lactation days 12-16 (altogether for 50-55 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. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. The dams were allowed to litter, and rear their offspring up to day 13 postpartum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (T4, TSH) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) 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. Based on macroscopic and histopathology findings at 300 mg/kg bw/day, pancreas of selected animals administered with 30 and 100 mg/kg bw/day doses were also processed and examined histologically. In addition, organs showing macroscopic findings at necropsy were also processed and examined histologically in single animals at 100 mg/kg bw/day (kidneys, pancreas and seminal vesicle and uterus). The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (PEG 400) only. Historical control data were also considered.

Results

Mortality

There was no mortality at 30, 100 or 300 mg/kg bw/day groups during the course of study.

Clinical observation

Adverse clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily or detailed weekly clinical observations or at the terminal functional observations. The behavior and physical condition of animals was not affected by the test item at any dose level (30, 100 or 300 mg/kg bw/day) during the entire observation period. Soft stool was detected in the bedding material in each cage of male and female animals of control, 30, 100 and 300 mg/kg bw/day.

Body weight and body weight gain

Test item related adverse changes in the body weight or body weight gain were not detected. The body weight development was not disturbed and it was comparable in the control and test item treated groups.

Food consumption

The mean daily food consumption was not affected by the test item in male or female animals at 30, 100 and 300 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods).

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (30, 100 and 300 mg/kg bw/day).

Reproductive performance

There were no test item related differences between the control and test item treated groups in the reproductive performance of male and female animals.

Delivery data of dams

There were no toxicologically relevant differences between the control and test item treated groups (30, 100 and 300 mg/kg bw/day) in the evaluated delivery parameters of dams.

Hematology

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 30, 100 or 300 mg/kg bw/day.

Clinical chemistry

Specific alterations related to test item effect were not detected in the examined clinical chemistry parameters at any dose level (male or female; 30, 100 or 300 mg/kg bw/day).

Serum thyroid hormones

There were no test item related changes in the serum thyroid hormone (T4, TSH) levels at any dose (parental male animals or PN13 offspring).

Necropsy

Adverse macroscopic alterations related to the effect of the test item were not found in the reproductive organs of male or female animals at 30, 100 or 300 mg/kg bw/day at the necropsy. Enlargement or paleness of pancreas was observed at 100 and 300 mg/kg bw/day in male animals in accordance with histopathological findings.

Organ weight

There were no test item related adverse changes in the weights (absolute and relative to body or brain weights) of selected organs in the animals at any dose level.

Histopathology

Histopathological examinations of the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 300 mg/kg bw/day. Increased amount of zymogen granules and flattened and basophilic acinar cells were observed in the pancreas in 1/1 male animal at 100 mg/kg bw/day and in 5/5 male and 3/5 female animals at 300 mg/kg bw/day. The cytomorphology of pancreas was normal in the selected animals of the low and mid dose groups. These findings might be indicative of a disorder in synthesis and secretion of the digestive enzymes and are associated with test item treatment.

Offspring

The offspring’s development was not adversely influenced at any dose level.

Under the conditions of the present study, Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyl- trimethoxysilane did not adversely influence the fertility or reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 30, 100 and 300 mg/kg bw/day doses administered by oral gavage. 300 mg/kg bw/day induced pancreatic changes: enlargement and pale color in male animals, increased amount of zymogen granules and flattened and basophilic acinar cells in male and female animals. The morphological change in the pancreas was considered test item related but its toxicological significance is equivocal because no related changes were detected in any of the examined parameters. Since neither inflammation, necrosis or apoptosis were observed nor an increase in severity over time compared to the 14 day-Dose Range-Finder study, reversibility of these exocrine pancreatic observations can be considered as very likely. The endocrine pancreas was not affected and cytomorphology of Langerhans-islets was normal in all cases. At 100 mg/kg bw/day, pancreatic changes (increased amount of zymogen granules and flattened and basophilic acinar cells) were observed in only one male animal. In conclusion, all observations in the pancreas are interpreted as non-adverse. 

There were no test item related changes in other animals administered with 100 mg/kg bw/day. There were no test item related changes in animals administered with 30 mg/kg bw/day.

The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams. The NOAEL for systemic toxicity of male/female rats was 300 mg/kg bw/day, the NOAEL for reproductive performance of male/female rats was 300 mg/kg bw/day and the NOAEL for F1 Offspring was 300 mg/kg bw/day, the highest dose tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Toxi-Coop ZRT, 8230 Balatonfüred, Arácsi út 97, Hungary
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane
EC Number:
924-669-1
Cas Number:
192526-20-8
Molecular formula:
No data available
IUPAC Name:
Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
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
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Male animals: 11-12 weeks (80-90 days) old
Female animals: 11-12 weeks (80-90 days) old
- Weight at study initiation:
Male animals: 349 – 397 g
Female animals: 187 – 235 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate cages
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females housed individually.
- Diet: ad libitum, 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
- Water: ad libitum, tap water
- Acclimation period: 20 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 6 mg/mL, 20 mg/mL and 60 mg/mL. Each formulation were prepared by careful weight measurement just prior to administration. Formulations were prepared daily and administered within a short period thereafter thus the stability of the formulations is considered to be not relevantly affected.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore PEG 400 will be used for preparing formulations appropriate for oral administration. PEG 400 is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 6 mg/mL, 20 mg/mL and 60 mg/mL
- Treatment volume: 5 mL/kg bw
- Lot/batch no.: 16I284004
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Five samples were taken (from different places) from each concentration (Groups 2, 3 and 4) and at least three parallels are measured twice during the study. Similarly, five samples were taken from the control solution (Group 1). Due to the solubility and instability feature of the test item, stability determination was not possible.
Duration of treatment / exposure:
Males dosed for at least 28 days (14 days pre-mating and 14 days mating plus an optional extended post-mating period until the necessary number of pregnant female animals is evident)
Females dosed for 14 days pre-mating, through 14 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose setting was based on findings obtained in a 14-day dose range finding study performed with Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane (study no. 666-400-0899, non-GLP). The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals.
Positive control:
none

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
Observations 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 behavior pattern, changes in gait, posture and response to handling. Special attention will be directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted on five male and five female animals randomly selected from each group during their respective last exposure week but before the blood sampling.

BODY WEIGHT: Yes
- Time schedule for examinations:
Parental males were weighed on the first day of dosing (day 0), at least weekly thereafter and at termination.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally measured on gestation days 10 in order to give accurate treatment volumes.

FOOD CONSUMPTION: Yes
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase as follows: premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals. Fasted body weight was determined for animals selected for toxicity examinations before the necropsy.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Anaesthetic used for blood collection: Yes, Isofluran anesthesia
- Animals fasted: Yes
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked: WBC (White Blood Cell leukocyte count), RBC (Red Blood Cell erythrocyte count), HGB (Hemoglobin concentration), HCT (Hematocrit, relative volume of erythrocytes), MCV (Mean Corpuscular erythrocyte Volume), MCH (Mean Corpuscular erythrocyte Hemoglobin), MCHC (Mean Corpuscular erythrocyte Hemoglobin Concentration), PLT (Platelet thrombocyte count), RET (Reticulocytes), Differential white blood cell count, APTT (Activated partial Thromboplastin Time), PT (Prothrombin Time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Animals fasted: Yes, Isofluran anesthesia
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked: ALT (Alanine Aminotransferase activity), AST (Aspartate Aminotransferase activity), TBIL (Total Bilirubin concentration), CREA (Creatinine concentration), UREA (Urea concentration), GLUC (Glucose concentration),CHOL (Cholesterol concentration), Na+ (Sodium concentration),K+ (Potassium concentration), ALB (Albumin concentration), TPROT (Total Protein concentration)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (T4,TSH) as follows:
- from all dams and at least two pups per litter on day 13 if feasible
- from all parent male animals at termination
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The appearance of the tissues and organs were observed, and any abnormality was recorded including details of the location, color, shape and size.
At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals was determined. In addition, for five males and females randomly selected from each group, adrenal glands,brain, heart, kidneys, liver, spleen and thymus was weighed. Absolute organ weight was reported. Relative organ weight (to body and brain weights) was calculated and reported.
The thyroid weight was determined after fixation.

HISTOPATHOLOGY: Yes
Detailed histological examination were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments ofthe ovary as well as the epithelial capsule and ovarian stroma.
In addition, these organs were processed and examined histologically in not mated, non-pregnant or not delivered females and males these females cohabited with in the low and middle dose groups.
Full histopathology examinations was performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. Histopathological examinations were performed for the pancreas in all dose groups.
Other examinations:
LITTER OBSERVATIONS:
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) presence of gross anomalies. Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete, post-natal day 0) and on postnatal day 4. The anogenital distance of each pup was determined on postnatal day 4. Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum T4 and TSH levels. The number of nipples/areolae in male pups was counted on postnatal day 13.

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Soft stool caused by the vehicle was detected in the bedding material in each cage: male and female animals of control, 30, 100 and 300 mg/kg bw/day groups from Day 2 up to the termination of the treatment. Protrusion (~6 mm) at the vagina connected with a tissue formation was observed for one female animal at 300 mg/kg bw/day between lactation days 0 and 15. This clinical sign was considered to be individual finding and not related to the treatment with the test item. There were no clinical signs in the other animals (male or female) of control, 30, 100 or 300 mg/kg bw/day groups, i.e. these animals exhibited normal behavior and physical condition with no abnormalities during the treatment period.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the control, 30, 100 or 300 mg/kg bw/day groups (male or female) during the course of study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight development was not adversely affected by the test item in male or female animals at 30, 100 or 300 mg/kg bw/day during the entire treatment period. There were no significant differences between the control and the test item treated male animals in the mean body weight although the mean body weight gain was significantly lower than in the control animals at 100 mg/kg bw/day between Days 13 and 20, and it was significantly higher than in the control group at 30 and 300 mg/kg bw/day between Days 41 and 48. There were no statistically significant differences between the control and test item treated groups in the mean body weight and mean body weight gain in female animals at 30, 100 and 300 mg/kg bw/day in the premating, gestation or lactation periods.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item or treatment related adverse changes in the food consumption of male and female animals at any dose level (30, 100 and 300 mg/kg bw/day).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, statistical significances were detected at the slightly lower mean red blood cell (erythrocyte) count (RBC) and mean hematocrit value at 300 mg/kg bw/day. In female animals, slightly but statistically significantly lower mean red blood cell (erythrocyte) count (RBC) was observed at 300 mg/kg bw/day. The statistically significant differences between the control and high dose treated groups for red blood cell parameters (RBC, HCT) were considered to have no toxicological relevance due to the minor degree and lack of any changes in other parameters. Furthermore, the values are still well within the historical control ranges, respectively.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, statistical significance was detected with respect to their control at the lower mean total bilirubin (TBIL) and mean creatinine concentration (CREA) at 300 mg/kg bw/day. These differences between control and test item treated male animals reached statistical significances however individual values were in line with the historical control and these changes were judged to have no toxicological relevance. All examined clinical chemistry parameters were comparable in female animals in the control and in all test item treated groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observations did not demonstrate any test item related adverse changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (30, 100 and 300 mg/kg bw/day, control).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals at 30 mg/kg bw/day, the mean brain weight (absolute) was slightly lower than in the control group. In male animals at 100 mg/kg bw/day, statistical significance with respect to the control was observed at the lower mean weights of brain (absolute and relative to body weight) and heart (absolute). At 300 mg/kg bw/day, the mean brain weights (absolute and relative to body weight) were lower and the body weight relative to brain weight and mean weight of epididymides relative to brain weight were slightly higher than in the control group in male animals. There were no statistically significant differences in female animals between the control and the 30, 100 or 300 mg/kg bw/day test item treated groups in the weights of the examined organs at the end of the treatment period. The changes in brain weights of male animals were with minor degree and not related to doses or histopathological alterations at 300 mg/kg bw/day. Change in epididymal weight was not associated with any pathological or histopathological findings. The slight change in heart weight was only detected in the mid dose treated male animals but not in the high dose. Therefore, these statistical significances in brain, heart and epididymides were considered to be toxicologically not relevant. Moreover, all values were well within historical control ranges of this rat strain.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item related changes were detected in the pancreas in male animals at 100 mg/kg bw/day (pale) and at 300 mg/kg bw/day (paleness or enlargement) at the necropsy being in full accordance with the histopathological findings. There were no macroscopic changes in the organs or tissues in male animals in the control and 30 mg/kg bw/day groups (12/12, both). At 100 mg/kg bw/day, the seminal vesicle (right side) was smaller than normal in two male animals (2/12). One of the latter also showed hydronephrosis of the right kidney with dilated ureter, larger than normal left kidney and pale pancreas (1/12). At 300 mg/kg bw/day, pale colored pancreas (8/12), enlarged pancreas (7/12) and thymic hemorrhage (1/12) were observed. There were no macroscopic findings in the organs or tissues in female animals in the control and 30 mg/kg bw/day groups (12/12, each). At 100 mg/kg bw/day, in a single female animal slight hydrometra was observed (1/12). At 300 mg/kg bw/day the vagina of one female animal (1/12) was thickened and extended on the uterine horns. This observation was considered as an individual disease and not caused by test item treatment. Hydronephrosis with ureter dilatation and – as a consequence – renal enlargement on the opposite site, smaller than normal seminal vesicle were individual disorders occurring in non-treated animals of this strain with similar age. In the lack of dose relevance – were present only in the mid dose male animals only – these were judged to be toxicologically not relevant. Hemorrhage in the thymus was probably due to circulatory disturbance developed during exsanguination procedure. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or another pathological lesion) these were judged not to be toxicologically
relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examinations did not reveal any test item related adverse alterations in the reproductive organs or tissues of male or female animals at 300 mg/kg/bw/day (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) Histological examination of the pancreas revealed increased amount of zymogen granules and flattened and basophilic acinar cells at 100 mg/kg bw/day (1/6 male) and at 300 mg/kg bw/day (5/5 male and 3/5 female) in mild or moderate degree. These findings were in accordance with the macroscopic changes in the pancreas (enlargement and paleness). No histopathological findings were reported in the pancreas in the low dose group (30 mg/kg bw/day). The increased amount of zymogen granules and the flattened and basophilic acinar cells – without acinar cell necrosis, atrophy or disorganization of acinar structure – was considered as histological signs of an adaptive response of the pancreatic acinar cells to test item treatment indicative of an adaptive acinar hypertrophy. These findings could be caused by an enhanced synthesis and secretion of proenzymes induced by the test item. The proenzymes are inactive precursors which do not induce adverse effects in acinar cells after prolonged storage. So, the correlation between the gross pathological findings and histological picture is obvious, but the toxicological significance is questionable. Since neither inflammation, necrosis or apoptosis were observed nor an increase in severity over time (refer to 14-day Dose Range Finding Study; Study no. 666-400-0899), reversibility of these exocrine pancreatic observations can be considered as very likely. Overall, these observations are likely to be interpreted adaptive changes induced by the test item and no adversity is to be expected. The endocrine pancreas was not affected and cytomorphology of Langerhans-islets was normal in all cases. In conclusion, all observations in the pancreas are interpreted as non-adverse.
In all male animals (12/12 control, 12/12 at 300 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism in all cases. Decreased amount of secrete in the seminal vesicle (one side) was observed in two male animals (2/2) at 100 mg/kg bw/day in accordance with macroscopic findings (smaller than normal). This finding was considered as individual disorder without toxicological relevance as there were no accompanying inflammatory or degenerative lesions. In the female animals (12/12 control, 11/12 at 300 mg/kg bw/day) the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. The cortical region of ovaries 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. In one female (1/1) at 300 mg/kg bw/day metritis was observed in the uterus. This finding was considered as individual disease, not related to the test item treatment. In one female animal (1/1) at 100 mg/kg bw/day dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.Alveolar emphysema in minimal or mild degree was observed in the lungs in the control (1/5 male and 2/5 dams) and at 300 mg/kg bw/day (1/5 male). Hyperplasia of bronchus associated lymphoid tissue (BALT) was noted for single animals in the control group (1/5 male, 1/5 dam). Acute hemorrhage was observed in the thymus in one male at 300 mg/kg bw/day (1/1). Severe hydronephrosis (one side) was observed in one male animal at 100 mg/kg bw/day (1/1). Alveolar emphysema in the lungs was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Hyperplasia of bronchus associated lymphoid tissue (BALT) is a physiological immune-morphological phenomenon, without toxicological significance. Thymic hemorrhage was a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the stomach, the small and large intestines, the liver, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the selected animals. The cyto-morphology of the endocrine glands was the same in the control and the treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The estrous cycle was not affected by the test item at any dose level (30, 100 or 300 mg/kg bw/day). There were no significant differences between the control and treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus during the pre-experimental or pre-mating period.
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, seminal vesicles, and coagulating glands was normal in all animals as well.
There were no significant differences between the control and test item treated (30, 100 and 300 mg/kg bw/day) groups in the copulatory and fertility indices (males and females) and in the gestation indices (females). The percentage of pregnant females, dams delivered, pregnants with liveborn(s) and the pre-coital interval and the mean number of conceiving days were comparable in female animals of control and test item treated animals.
Details on results:
Delivery Data of Dams
There were no relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (30, 100 or 300 mg/kg bw/day). The mean number of implantation sites per dams and the mean of postimplantation loss were comparable 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 on post-partal day 0, in the percentage of dams with viable offspring on postpartal day 0 or in the live birth index.

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyl- trimethoxysilane did not adversely influence the fertility or reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 30, 100 and 300 mg/kg bw/day doses administered by oral gavage. The NOAEL for systemic toxicity of male/female rats was 300 mg/kg bw/day, the NOAEL for reproductive performance of male/female rats was 300 mg/kg bw/day and the NOAEL for F1 Offspring was 300 mg/kg bw/day, the highest dose tested.
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422 was performed to obtain initial information on the toxic potential of Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane 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 13 post- partum when repeatedly administered orally (by gavage) to parental animals at doses of 30 mg/kg bw/day, 100 mg/kg bw/day and 300 mg/kg bw/day compared to control animals. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 30, 100 and 300 mg/kg bw/day doses corresponding to concentrations of 0, 6, 20 and 60 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. Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane concentrations in the dosing formulations varied in the acceptable range between 101 % and 109 % 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 50 days). Females were additionally exposed through the gestation period and up to lactation days 12-16 (altogether for 50-55 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. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. The dams were allowed to litter, and rear their offspring up to day 13 postpartum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (T4, TSH) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) 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. Based on macroscopic and histopathology findings at 300 mg/kg bw/day, pancreas of selected animals administered with 30 and 100 mg/kg bw/day doses were also processed and examined histologically. In addition, organs showing macroscopic findings at necropsy were also processed and examined histologically in single animals at 100 mg/kg bw/day (kidneys, pancreas and seminal vesicle and uterus). The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (PEG 400) only. Historical control data were also considered.

Results

Mortality

There was no mortality at 30, 100 or 300 mg/kg bw/day groups during the course of study.

Clinical observation

Adverse clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily or detailed weekly clinical observations or at the terminal functional observations. The behavior and physical condition of animals was not affected by the test item at any dose level (30, 100 or 300 mg/kg bw/day) during the entire observation period. Soft stool was detected in the bedding material in each cage of male and female animals of control, 30, 100 and 300 mg/kg bw/day.

Body weight and body weight gain

Test item related adverse changes in the body weight or body weight gain were not detected. The body weight development was not disturbed and it was comparable in the control and test item treated groups.

Food consumption

The mean daily food consumption was not affected by the test item in male or female animals at 30, 100 and 300 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods).

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (30, 100 and 300 mg/kg bw/day).

Reproductive performance

There were no test item related differences between the control and test item treated groups in the reproductive performance of male and female animals.

Delivery data of dams

There were no toxicologically relevant differences between the control and test item treated groups (30, 100 and 300 mg/kg bw/day) in the evaluated delivery parameters of dams.

Hematology

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 30, 100 or 300 mg/kg bw/day.

Clinical chemistry

Specific alterations related to test item effect were not detected in the examined clinical chemistry parameters at any dose level (male or female; 30, 100 or 300 mg/kg bw/day).

Serum thyroid hormones

There were no test item related changes in the serum thyroid hormone (T4, TSH) levels at any dose (parental male animals or PN13 offspring).

Necropsy

Adverse macroscopic alterations related to the effect of the test item were not found in the reproductive organs of male or female animals at 30, 100 or 300 mg/kg bw/day at the necropsy. Enlargement or paleness of pancreas was observed at 100 and 300 mg/kg bw/day in male animals in accordance with histopathological findings.

Organ weight

There were no test item related adverse changes in the weights (absolute and relative to body or brain weights) of selected organs in the animals at any dose level.

Histopathology

Histopathological examinations of the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 300 mg/kg bw/day. Increased amount of zymogen granules and flattened and basophilic acinar cells were observed in the pancreas in 1/1 male animal at 100 mg/kg bw/day and in 5/5 male and 3/5 female animals at 300 mg/kg bw/day. The cytomorphology of pancreas was normal in the selected animals of the low and mid dose groups. These findings might be indicative of a disorder in synthesis and secretion of the digestive enzymes and are associated with test item treatment.

Offspring

The offspring’s development was not adversely influenced at any dose level.

Under the conditions of the present study, Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyl- trimethoxysilane did not adversely influence the fertility or reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 30, 100 and 300 mg/kg bw/day doses administered by oral gavage. 300 mg/kg bw/day induced pancreatic changes: enlargement and pale color in male animals, increased amount of zymogen granules and flattened and basophilic acinar cells in male and female animals. The morphological change in the pancreas was considered test item related but its toxicological significance is equivocal because no related changes were detected in any of the examined parameters. Since neither inflammation, necrosis or apoptosis were observed nor an increase in severity over time compared to the 14 day-Dose Range-Finder study, reversibility of these exocrine pancreatic observations can be considered as very likely. The endocrine pancreas was not affected and cytomorphology of Langerhans-islets was normal in all cases. At 100 mg/kg bw/day, pancreatic changes (increased amount of zymogen granules and flattened and basophilic acinar cells) were observed in only one male animal. In conclusion, all observations in the pancreas are interpreted as non-adverse.  There were no test item related changes in other animals administered with 100 mg/kg bw/day. There were no test item related changes in animals administered with 30 mg/kg bw/day.

The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams. The NOAEL for systemic toxicity of male/female rats was 300 mg/kg bw/day, the NOAEL for reproductive performance of male/female rats was 300 mg/kg bw/day and the NOAEL for F1 Offspring was 300 mg/kg bw/day, the highest dose tested.