reaction product of diphenylmethanediisocyanate, toluenediisocyanate ( reaction mass of isomers: 65 % 2,4- and 35 % 2,6-diisocyanate), octylamine, oleylamine and 4-ethoxyaniline (molar ratio 3.88:1:6.38:0.47:2.91)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A study according OECD TG 422 was performed. 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), 100, 300 and 1000 mg/kg bw/day doses. The substance did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male or female Han:WIST rats at 100, 300 or 1000 mg/kg bw/day (by oral gavage), as investigated in this study. There were no signs of systemic toxicity in selected male or female animals at 100, 300 or 1000 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.

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

NOAEL for systemic toxicity

of male/ female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance

of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 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:

November 2019 - March 2020

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:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 885.3650 (Microbial Pesticide, Reproductive/Fertility Effects)

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

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
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 12 wks
- Weight at study initiation: Male animals: 332 – 381 g; Female animals: 200 – 242 g
- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females will be housed individually.
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice
- Water: ad libitum, tap water
- Acclimation period: 20

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

Route of administration:

oral: gavage

Vehicle:

other: 1 % aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for four days and stored at 5 ± 3 °C until use.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water. Therefore, 1 % methylcellulose was used for preparing formulations appropriate for oral administration. 1 % methylcellulose is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 20 mg/mL, 60 mg/mL and 200 mg/mL
- Treatment volume: 5 mL/kg bw

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 successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 100 % at ca. 1 mg/mL and 106 % at ca. 200 mg/mL).

Duration of treatment / exposure:

Males were 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); then they were sacrificed.
Females were 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 up to the day before sacrifice.

Frequency of treatment:

daily

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

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 Urea 6 (non-GLP; study no. 674-400-4426).

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 condu
cted 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 of the 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):

Dermal changes – alopecia and scars on the skin – were observed in single male and female animals independently from the test item. Alopecia and scars on the skin are common findings in rats of this strain occurring also in not treated animals.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in control or test item administered (100, 300 or 1000 mg/kg bw/day) groups (male or female) during the course of study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was not affected by the test item in male or female animals administered with 100, 300 or 1000 mg/kg bw/day.
The mean body weight and body weight gain were comparable to their control in male and female animals at 100, 300 and 1000 mg/kg bw/day during the entire observation period (pre-mating, mating and post-mating periods in male animals, pre-mating, gestation and lactation periods in female animals).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was not affected in male or female animals at 100, 300 and 1000 mg/kg bw/day.
The mean daily food consumption of male and female animals was comparable with their control at 100, 300 and 1000 mg/kg bw/day during the entire observation period (pre-mating, gestation and lactation periods).

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):

There were no test item related changes in the examined hematological or blood coagulation parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day.
The examined hematological and blood coagulation parameters were comparable to their control in male animals at 100, 300 and 1000 mg/kg bw/day groups and in female animals at 100 and 300 mg/kg bw/day groups.
In the female animals, statistical significance was detected at the slightly higher mean corpuscular hemoglobin content (MCH) at 1000 mg/kg bw/day when compared to the control. All individual values of MCH except two met well the historical control values and there were no related changes in the other hematological parameters. Therefore, the differences were considered to have no toxicological relevance

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examined clinical chemistry parameters were not adversely affected in male and female animals at 100, 300 or 1000 mg/kg bw/day.
Statistical significance with respect to the control was observed at the slightly lower mean enzyme activity of alanine aminotransferase (ALT) in male animals at 300 and 1000 mg/kg bw/day.
In the female animals, the mean concentrations of urea (300 mg/kg bw/day) and creatinine (100, 300 and 1000 mg/kg bw/day) were lower than in the control group.
These minor, statistically significant differences with respect to the control were of no toxicological relevance in the lack of dose dependency and related changes in other clinical pathology parameters or organ pathology. Moreover, the individual values remained well within the historical control range except for creatinine concentrations of three female animals in the control group.

Endocrine findings:

no effects observed

Description (incidence and severity):

The thyroid hormone levels (FT3, FT4 and TSH) were not adversely affected in the parental male animals or in PN13 offspring at any dose levels.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observations did not demonstrate any test item related changes in selected animals. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (control, 100, 300 and 1000 mg/kg bw/day; n=5 animals/sex/group).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological investigations did not reveal any toxic or other test item related lesions detectable by microscopic examination of the investigated reproductive and other organs of male and female animals at 1000 mg/kg bw/day.
In the examined male animals (12/12 control, 1/1 at 100 mg/kg bw/day, 1/1 at 300 mg/kg bw/day, 12/12 at 1000 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histological normal and characteristic on the sexually mature organism in all cases.
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, prostate, seminal vesicles, and coagulating glands was normal in all cases as well (12/12 control, 1/1 at 100 mg/kg bw/day, 1/1 at 300 mg/kg bw/day, 12/12 at 1000 mg/kg bw/day).
In the female animals – 11/11 dams and 1/1 not-delivered pregnant female in control group, 1/1 not mated female at 100 mg/kg bw/day, 1/1 non-pregnant female at 300 mg/kg bw/day, 12/12 dams at 1000 mg/kg bw/day –– the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle. 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.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals. In not delivered pregnant female animal in the control group (1/12), dilatation of uterine horn and metritis – in connection with the intrauterine death of fetuses – is considered as an individual disease.
The dilatation of uterine horns in single animals at 100 mg/kg bw/day (1/1 not mated), at 300 mg/kg bw/day (1/1 non-pregnant) and at 1000 mg/kg bw/day (1/12 dam) is a slight neuro-hormonal phenomenon in connection with the sexual function – proestrus phase – of the genital organs.
One or both sided pyelectasia in the kidneys (1/6 male control; 2/2 male at 100 mg/kg bw/day; 2/2 male and 1/1 female at 300 mg/kg bw/day; 1/6 male and 2/5 female at 1000 mg/kg bw/day) without degenerative, inflammatory or other histological (fibrotic etc.) lesion is considered as a common finding in laboratory rats without toxicological significance.
Atrophy of hair follicles accompanied with focal or multifocal alopecia (1/1 male at 100 mg/kg bw/day, 1/5 dam in control group and 1/5 dam at 1000 mg/kg bw/day) or cystadenoma (1/1 male at 100 mg/kg bw/day) are common findings in laboratory rats.
No morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The examined parameters of the estrous cycle were comparable in the control and test item administered animals (100, 300 and 1000 mg/kg bw/day).
There were no statistically significant differences between the control and test item treated groups in the number or percentage of animals with regular cycles, in the mean number or length of cycles, mean number of days in pro-estrus, estrous or diestrus during the pre-experimental periods.
Statistically significant difference with respect to the control was observed at the slightly lower mean number of days in pro-estrous in female animals at 300 mg/kg bw/day during the pre-mating period. The values were well in the historical control range; therefore, this minor difference was considered to be indicative of biological variation and no dose dependency was present.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In the examined male animals (12/12 control, 1/1 at 100 mg/kg bw/day, 1/1 at 300 mg/kg bw/day, 12/12 at 1000 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histological normal and characteristic on the sexually mature organism in all cases.
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, prostate, seminal vesicles, and coagulating glands was normal in all cases as well (12/12 control, 1/1 at 100 mg/kg bw/day, 1/1 at 300 mg/kg bw/day, 12/12 at 1000 mg/kg bw/day).

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not influenced by the treatment with the test item in male or female animals at 100, 300 or 1000 mg/kg bw/day.
Statistical significance with respect to the control was observed at the lower percentage of mated animals (copulatory index) at 100 mg/kg bw/day and at the lower percentage of fertile animals (fertility index) at 300 mg/kg bw/day (male and female). Concurrently, statistical significance was also noted for the higher percentage of infertile male animals and higher percentage of non-pregnant female animals at 300 mg/kg bw/day.
The gestation index (percentage of dams delivered) exceeded the control in all test item groups because one pregnant female animal did not give birth in the control group.
These statistically significant differences were indicative of biological variation and not related to the test item. Changes in the reproductive indices were only detected in animals at 100 or 300 mg/kg bw/day but not at the higher dose group and values met well the historical control.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related clinical signs in the offspring from post-natal day 0 to 13. The body temperature was judged to be cold in some pups at 300 mg/kg bw/day (9/122) and at 1000 mg/kg bw/day (14/123). No milk was detected in the stomach in some offspring (1/122 at 300 mg/kg bw/day, 12/123 at 1000 mg/kg bw/day). One pup at 300 mg/kg bw/day (1/122) was cyanotic for one day. Alopecia on the whole body was observed in all pups of two litters – in one dam of the control and in one dam of 1000 mg/kg bw/day groups (n=8 pups in both litters). The signs were observed with low incidence, which met well the historical control. Therefore, these signs were considered to be toxicologically not relevant.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality. The extrauterine mortality was low and comparable in the control, 100, 300 and 1000 mg/kg bw/day groups from birth (post-natal day 0) to post-natal day 13. The mean number of live-born per litter, and number of viable pups per litter were comparable in the control and 100, 300 and 1000 mg/kg bw/day groups on postnatal days 0, 4 and 13. There were no significant differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development of the offspring was undisturbed at 100, 300 and 1000 mg/kg bw/day between post-natal days 0 and 13. The mean litter weights and the mean litter weight gain were similar in the control and at 100, 300 or 1000 mg/kg bw/day groups on postnatal days 0, 4 and 13. There was no difference between the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) in the mean pup weight and pup weight gain per litter on post-natal days 0, 4 and 13.
Considering the offspring’s body weight in males and females separately, statistically significant difference with respect to the control was detected at the slightly higher mean weight of male pup at 100 mg/kg bw/day on PN 4. This minor difference with respect to the control was considered to be toxicologically not relevant due to the minor degree and lack of dose relevance.

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

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distances were not affected by the test item at 100, 300 and 1000 mg/kg bw/day in male or female offspring. Statistical significances were noted for the slightly longer mean absolute and normalized anogenital distances in male and offspring at 100 mg/kg bw/day comparing to their control. These differences were with minor degree and still within the range of the historical control data and similar finding was not detected in pups of the high dose group. Therefore, they were considered to have no toxicological relevance. The mean absolute and normalized anogenital distances were comparable in female and offspring in the control, 100, 300 and 1000 mg/kg bw/day groups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on post-natal day 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination. In dead pup (1/1) in control group swelling on the abdomen (0.5 cm) was detected at necropsy on PN10. There were no macroscopic findings in offspring subjected to early necropsy: 1/3 dead at 300 mg/kg bw/day; and 2/2 stillborn (negative lung flotation test). Two dead pups at 300 mg/kg bw/day (2/3) was partially cannibalized.
Both sides pyelectasia was noted for one female pup at 300 mg/kg bw/day group (1/22). Alopecia on the whole body was observed in offspring in control (2/22) and at 1000 mg/kg bw/day (2/24) groups.
Pyelectasia and alopecia are species specific alterations in offspring of this strain. Histopathological examinations supported these findings: Mild atrophy of hair follicles in the skin of offspring with alopecia (2/2 control and 2/2 at 1000 mg/kg bw/day) and pyelectasia (1/1) without degenerative, inflammatory or other histological (fibrotic etc.) lesion were detected at the microscopic investigation.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The sex distribution of male and female pups (mean percentage of male pups per litter) was comparable in all groups on post-natal days 0, 4 and 13. There were no significant differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the conditions of the present study, Urea 6 did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male or female Han:WIST rats at 100, 300 or 1000 mg/kg bw/day (by oral gavage), as investigated in this study.
There were no signs of systemic toxicity in selected male or female animals at 100, 300 or 1000 mg/kg bw/day.
The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/ female rats: 1000 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day

Executive summary:

The purpose of this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was to obtain initial information on the toxic potential of Urea 6 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 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day compared to control animals according to OECD 422.

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), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, 1 % aqueous methylcellulose.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity).

The concentration of the test item in the dosing formulations used for animal’s treatment was checked two times during the study. Concentration of Urea 6 in the dosing formulations varied between the range of 98 % and 104 % of the nominal values and formulations were homogenous thereby 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 42 days). Dams were additionally exposed through the gestation period and up to lactation days 12-15 (altogether for 51-54 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, blood coagulation, 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 and on the day of the necropsy.

The dams were allowed to litter and rear their offspring up to day 13 post-partum or shortly thereafter. 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 determination of serum levels of thyroid hormones (FT3, FT4, TSH) from 2-6 pups per litter (in litters with 9 or more pups) on post-natal day 4, from all dams and from 2-7 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.

Additionally, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed for five male and five female animals randomly selected from each group.

Histopathology examination was performed on reproductive organs (testes, epididymides, prostate, and seminal vesicles with coagulating gland, uterus with oviduct, cervix, vagina and ovaries) in the control and high dose groups and in not mated and non-pregnant female animals and the male these females cohabited with in the low (100 mg/kg bw/day) or mid (300 mg/kg bw/day).

Full histopathology examinations were performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups.

In addition, organs or tissues showing macroscopic changes (kidneys, skin) were also processed and examined histologically in animals in control, low or mid or high (1000 mg/kg bw/day) dose groups.

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle only. Historical control data were also considered.

There was no test item related mortality in 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of study.

Adverse signs of systemic toxicity related to the test item were not detected at any dose level (100, 300 or 1000 mg/kg bw/day, male or female) at the daily or detailed weekly clinical observations or at the terminal functional observations.

The mean body weight gain and mean body weight were not influenced by the test item in male or female animals administered with 100, 300 or 1000 mg/kg bw/day.

The food consumption was not adversely affected in male or female animals at 100, 300 or 1000 mg/kg bw/day.

A test item influence on the estrous cycle was not detected at any dose level (100, 300 or 1000 mg/kg bw/day).

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

Delivery data of dams was not adversely affected at 100, 300 or 1000 mg/kg bw/day dose level.

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day.

The examined clinical chemistry parameters were not affected in male and female animals at 100, 300 or 1000 mg/kg bw/day.

The FT3 and FT4 thyroid hormone levels were not adversely affected in the parental male animals or in PN13 offspring at any dose levels.

The TSH concentrations were below the detection limit in parental male animals (0, 100, 300 and 1000 mg/kg bw/day), so it was considered to be in the physiological range of this strain, sex and age, as reflected by the historical control data. The TSH concentrations were comparable in the PN13 offspring in the control and test item treated groups.

Macroscopic alterations related to the effect of the test item were not detected in male or female animals at 100, 300 or 1000 mg/kg bw/day at the terminal necropsy.

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes, epididymides and seminal vesicles of male animals at any dose level.

Histopathological examinations of the investigated sexual organs and accessory sex organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day or in non-pregnant female animal or in its male partner at 300 mg/kg bw/day.

There were no test item related lesions detectable by microscopic (histopathological) examination of the investigated organs of selected male and female animals at 1000 mg/kg bw/day.

The offspring’s development was not adversely influenced at any dose level. No effects on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

Under the conditions of the present study, Urea 6 did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male or female Han:WIST rats at 100, 300 or 1000 mg/kg bw/day (by oral gavage), as investigated in this study.

There were no signs of systemic toxicity in selected male or female animals at 100, 300 or 1000 mg/kg bw/day.

The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.

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

NOAEL for systemic toxicity  of male/ female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance  of male/ 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:

Reliable study according to OECD Guideline and in compliance with GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The purpose of this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was to obtain initial information on the toxic potential of Urea 6 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 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day compared to control animals according to OECD 422.

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), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, 1 % aqueous methylcellulose.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity).

The concentration of the test item in the dosing formulations used for animal’s treatment was checked two times during the study. Concentration of Urea 6 in the dosing formulations varied between the range of 98 % and 104 % of the nominal values and formulations were homogenous thereby 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 42 days). Dams were additionally exposed through the gestation period and up to lactation days 12-15 (altogether for 51-54 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, blood coagulation, 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 and on the day of the necropsy.

The dams were allowed to litter and rear their offspring up to day 13 post-partum or shortly thereafter. 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 determination of serum levels of thyroid hormones (FT3, FT4, TSH) from 2-6 pups per litter (in litters with 9 or more pups) on post-natal day 4, from all dams and from 2-7 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.

Additionally, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed for five male and five female animals randomly selected from each group.

Histopathology examination was performed on reproductive organs (testes, epididymides, prostate, and seminal vesicles with coagulating gland, uterus with oviduct, cervix, vagina and ovaries) in the control and high dose groups and in not mated and non-pregnant female animals and the male these females cohabited with in the low (100 mg/kg bw/day) or mid (300 mg/kg bw/day).

Full histopathology examinations were performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups.

In addition, organs or tissues showing macroscopic changes (kidneys, skin) were also processed and examined histologically in animals in control, low or mid or high (1000 mg/kg bw/day) dose groups.

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle only. Historical control data were also considered.

There was no test item related mortality in 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of study.

Adverse signs of systemic toxicity related to the test item were not detected at any dose level (100, 300 or 1000 mg/kg bw/day, male or female) at the daily or detailed weekly clinical observations or at the terminal functional observations.

The mean body weight gain and mean body weight were not influenced by the test item in male or female animals administered with 100, 300 or 1000 mg/kg bw/day.

The food consumption was not adversely affected in male or female animals at 100, 300 or 1000 mg/kg bw/day.

A test item influence on the estrous cycle was not detected at any dose level (100, 300 or 1000 mg/kg bw/day).

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

Delivery data of dams was not adversely affected at 100, 300 or 1000 mg/kg bw/day dose level.

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day.

The examined clinical chemistry parameters were not affected in male and female animals at 100, 300 or 1000 mg/kg bw/day.

The FT3 and FT4 thyroid hormone levels were not adversely affected in the parental male animals or in PN13 offspring at any dose levels.

The TSH concentrations were below the detection limit in parental male animals (0, 100, 300 and 1000 mg/kg bw/day), so it was considered to be in the physiological range of this strain, sex and age, as reflected by the historical control data. The TSH concentrations were comparable in the PN13 offspring in the control and test item treated groups.

Macroscopic alterations related to the effect of the test item were not detected in male or female animals at 100, 300 or 1000 mg/kg bw/day at the terminal necropsy.

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes, epididymides and seminal vesicles of male animals at any dose level.

Histopathological examinations of the investigated sexual organs and accessory sex organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day or in non-pregnant female animal or in its male partner at 300 mg/kg bw/day.

There were no test item related lesions detectable by microscopic (histopathological) examination of the investigated organs of selected male and female animals at 1000 mg/kg bw/day.

The offspring’s development was not adversely influenced at any dose level. No effects on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

Under the conditions of the present study, Urea 6 did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male or female Han:WIST rats at 100, 300 or 1000 mg/kg bw/day (by oral gavage), as investigated in this study.

There were no signs of systemic toxicity in selected male or female animals at 100, 300 or 1000 mg/kg bw/day.

The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.

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

NOAEL for systemic toxicity  of male/ female rats: 1000 mg/kg bw/day

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

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

Effects on developmental toxicity

Description of key information

A study according OECD TG 422 was performed. 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), 100, 300 and 1000 mg/kg bw/day doses. The substance did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male or female Han:WIST rats at 100, 300 or 1000 mg/kg bw/day (by oral gavage), as investigated in this study. There were no signs of systemic toxicity in selected male or female animals at 100, 300 or 1000 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.

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

NOAEL for systemic toxicity

of male/ female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance

of male/ female rats: 1000 mg/kg bw/day

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable study according to OECD Guideline 422 and in compliance with GLP.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the test results, no classification and labelling is required according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.