reaction product of diphenylmethanediisocyanate, octylamine, 4-ethoxyaniline and ethylenediamine (1:0,37:1,53:0,05)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-15 to 2017-04-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550 “Reproduction / Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Premating exposure duration for parental (P0) animals: 2 weeks
- Basis for dose level selection: The dose setting is based on findings obtained in an OECD 421 study performed with a structural analogue of Polyurea 4 (study no. 674.421.3886, GLP, CAS 430-940-0). Doses were selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
- Route of administration: oral

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-750-8

EC Name:

-

Cas Number:

1266545-86-1

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Reaction product of (C8 – C18) aliphatic primary amines, ethylene diamine, p-phenetidine with 4,4’-methylenediphenyl diisocyanate

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for reproduction toxicity 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., Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Male animals: 89 – 91 days old, Female animals: 89 – 91 days old
- Weight at study initiation: Male animals: 325 – 409 g, Female animals: 200 – 245 g
The weight variation in animals involved in the study did not exceed 20 % of the mean group weight of each sex on Day 0.
- Housing: Before mating: 2 animals of the same sex/cage, Mating: 1 male and 1 female/cage, Mated females were housed individually, Males after mating: 2 animals/cage
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, Soest, Germany, ad libitum
- Water: tap water ad libitum
- Acclimation period: 34 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 5 % Tween 80 in distilled water

Details on exposure:

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

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore 5 % Tween 80 was used for preparing formulations appropriate for oral administration. 5 % Tween 80 was a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 20, 60 and 200 mg/L corresponding to 100, 300 and 1000 mg/kg bw/day
- Amount of vehicle: 5 mL dose preparation/kg body weight

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation occurred
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single housing

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations (checking of concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility. The test substance was determined by a reverse phase HPLC method with UV detection on a Hypersil BDS C18 column.
Ten samples (0.5 mL, each) were taken from different places from each concentration (Groups 2, 3 and 4) and all samples were measured. Similarly, ten samples (0.5 mL, each) were taken from the vehicle, from different places, (Group 1) and five sample was analyzed. Concentration of the test item in the dosing formulations varied in the range of 99 and 108 % of the nominal values at both analytical occasions.

Duration of treatment / exposure:

Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. The mating phase started after 14-days of pre-mating. Male animals were dosed for 55 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 12 - 18 (altogether for 55 days).
Non-pregnant and not delivered female animals were treated up to and including the day before necropsy (altogether for 55 days).

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

vehicle control

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

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 an OECD 421 study performed with a structural analogue (study no. 674.421.3886, GLP CAS 430-940-0). The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect.

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, runts, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
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).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals one day after the last treatment.
- Maternal animals: All surviving animals one day after the last treatment.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examinations were performed on the ovaries, testes and epididymides of all animals in the control and high dose groups. In addition, these organs were processed and examined histologically in non-pregnant or not delivered females and males, which were cohabited with these females in the low and middle dose groups: one non pregnant female at 100 mg/kg bw/day (1/1; and its male partner). The uterus with macroscopic finding (hydrometra) was also processed in one control dam (1/10), in two dams at 100 mg/kg bw/day (2/11), in two dams at 300 mg/kg bw/day (2/12) and in one of the non-pregnant female animals at 1000 mg/kg bw/day (1/2). Seminal vesicle with coagulating gland was examined histologically in one male animal of 300 mg/kg bw/day based on macroscopic observation (smaller than normal).

Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. The thyroid glands of all males and females were preserved. Thyroid and parathyroid were preserved together with the larynx. 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 were determined. Absolute organ weight was reported. Relative organ weights (to body and brain weight) were calculated and reported.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.

GROSS NECROPSY
- Gross necropsy consisted of examinations for external abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands of one male and one female pup per litter were preserved.

Statistics:

The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.

Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.

The frequency of clinical signs, pathology and histopathology findings were calculated.

Results were evaluated in comparison with values of control group (i.e. control value).

Reproductive indices:

For males and females: Copulatory Index, Fertility Index,
For females: Gestation Index

Offspring viability indices:

Post-implantation mortality, Post-natal mortality, Survival Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Scars were detected on the neck and under the ear in few male animal: on the neck at 300 mg/kg bw/day (1/12) from Day 12 up to the end of the study; on the neck at 1000 mg/kg bw/day (1/12) between Days 10 and 26; as well as under the ear at 1000 mg/kg bw/day (1/12) from Day 34 up to the termination of the study. Scars are a common spontaneous finding in this strain of experimental rats and is seen also in untreated rats therefore was considered to be toxicologically not relevant.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal at 1000 mg/kg bw/day was over anesthetized by mistake at the blood sampling for determination of T4 serum levels and was subjected to necropsy on Lactation day 13. There were no preceding clinical signs, body weight changes or macroscopic changes in the organs or tissues at the necropsy in this animal.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A decreased amount of secretum in the seminal vesicle was observed in one male animal at 300 mg/kg bw/day (1/1), which was considered to be an individual finding. In some female animals (1/1 control dam; 2/2 dams at 100 mg/kg bw; 2/2 dams at 300 mg/kg bw/day), dilatation of uterine horns was observed without inflammatory or degenerative lesions and in connection with the macroscopically detected hydrometra. This phenomenon was considered as a physiological finding in connection with the normal sexual cycle of uterus (pro-estrous phase).

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical significances with respect to the relevant control were noted for a higher fertility index at 300 mg/kg bw/day the due to the higher percentage of fertile male animals at 300 mg/kg bw/day.
Compared to the concurrent control groups, statistical significances were noted for the higher fertility index at 300 mg/kg bw/day and for the higher gestation indices at and 100, 300 and 1000 mg/kg bw/day in female animals.
These slight, but statistically significant differences in the parameters above were considered to be indicative of biological variation due to the lack of a clear and consistent dose-response-relations ship.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test-item related adverse effects observed

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test-item related adverse effects observed

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of dead pups was slightly higher in the control group than that in the test item treated groups with statistical significance at 1000 mg/kg bw/day indicating biological variation.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical significance was detected with respect to the control at the slightly higher mean pup weights at 100 and 1000 mg/kg bw/day in male pups and at 100, 300 and 1000 mg/kg bw/day in female pups on postnatal day 4. These slight differences were not considered to be related to the test item due to the minor degree and in the 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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and normalized in male or female offspring) or nipple retention (male) were not affected by the treatment.
The absolute anogenital distance was slightly but statistically significantly longer in male pups of the test item treated dams at 1000 mg/kg bw/day with respect to the control in full compliance with the mean body weight of these pups. However there was no difference in the normalized anogenital distance between these groups.
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 postnatal day 13.
In the female offspring, statistical significance was detected at the slightly shorter anogenital distances (absolute and normalized) at 100, 300 and 1000 mg/kg bw/day with respect to their control. In the lack of dose relevance and due to the minor degree, this slight difference with respect to the control was not considered to be toxicologically important.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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: no adverse effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Levels (NOAEL) for systemic toxicity and reproductive toxicity was determined to be 1000 mg/kg bw as no adverse effects were observed.

Executive summary:

The subject of this study was the Reproduction/Developmental Toxicity Screening Test with the test item in the rat according to OECD 421. Four groups of Hsd.Han: Wistar 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 corresponding to concentrations of 0, 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw. Control animals only received the vehicle (5 % Tween 80 in distilled water). 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 55 days). Females were additionally exposed through the gestation period and up to lactation days 12 - 18, i.e. up to the day before necropsy (altogether for 55 days). The dams were allowed to litter, and rear their offspring at least up to day 13 postpartum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter. There was no test item related mortality at any dose level.

Adverse signs of systemic toxicity related to the test item were not detected at any dose level, test item related changes in the body weight or body weight gain or organ weights were not detected. A test item influence on the estrous cycle was not detected at any dose level. There were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals. Histopathological examinations of male and female genital organs (ovaries, testes and epididymides) did not reveal any test item related changes. No adverse effect on the mortality, clinical signs or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected. The No Observed Adverse Effect Levels (NOAEL) for systemic toxicity and reproductive toxicity was determined to be 1000 mg/kg bw as no adverse effects were observed.