1,3-divinylimidazolidin-2-one

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-22 - 2015-03-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD Guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3-divinylimidazolidin-2-one

EC Number:

237-457-2

EC Name:

1,3-divinylimidazolidin-2-one

Cas Number:

13811-50-2

Molecular formula:

C7H10N2O

IUPAC Name:

1,3-diethenylimidazolidin-2-one

Details on test material:

- Name of test material (as cited in study report): 1,3-Dinvinylimidazolodin-2-on
- Test substance No.: 14/450-1
- Physical state: Colourless to yellow solid
- Analytical purity: 99,8 corrected area %
- Lot/batch No.: P53/14
- Expiration date of the lot/batch: 11 August 2015
- Stability under test conditions: stable
- Storage condition of test material: At room temperature

Test substance handling: No specific handling conditions required

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 313 g (males), 212 g (females)
- Fasting period before study: none
- Housing: groups of 5 animals/sex/cage (premating males and females and postmating males), otherwise single cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) are be prepared daily within 6 hours prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is insoluble in water, but soluble in corn oil
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

29 days (males), 40 - 45 days (females)

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150 and 450 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on a 14-day dose range finding study in which dose levels of 100, 300 and 1000 mg/kg bw/day
were tested and no adverse findings were seen at 100 mg/kg bw/day. At the higher dose levels clinical signs of toxicity, reduced body weight gain
and food consumption, and changes in clinical biochemistry values and organ weights were noted in a dose-related manner

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all
animals. Once prior to start of treatment and at weekly intervals during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed
on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
Feed consumption was measured per cage over weekly intervals during the study, with exemption of the mating period, during which no feed
consumption was registered.

GENERAL REPRODUCTION DATA.
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of
pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was
examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest,
pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

HAEMATOLOGY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
White blood cells
Differential leukocyte count 8neutrophils, lymphocytes, monocytes, eosinophils, basophils9
Red blood cells
Reticulocytes
Red blood cell distribution width
Haemoglobin
Haematocri
Mean corpuscular volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
Platelets

Clotting Potential
Prothrombin time
Activated Partial thromboplastin time

CLINICAL CHEMISTRY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
Alanine aminotransferase
Aspartartate aminotransferase
Alkaline phosphatase
Total Protein
Albumin
Total Bilirubin
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate
Bile acids

Furthermore, from the selected 5 animals/sex/group an additional blood sample (1.2 mL) was collected from the retro-orbital sinus into serum tubes for measurement of thyroid-stimulating hormone (TSH) and the thyroid hormones triiodothyronine (T3) and thyroxine (T4). After clotting and
centrifugation, serum samples were stored at ≤-75°C pending analysis within 2 months after collection

Thyroid hormone analyses:
Thyroid stimulating hormone (TSH)
total triiodothyronine (T3)
total thyroxine (T4)

NEURO-BEHAVIOURAL TESTING (FOB) AND SPONTANEOUS MOTOR ACTIVITY
During neuro-behavioural testing, the observer was unaware of the treatment of the animals. FOB and spontaneous motor activity were assessed in
all study animals during the predose phase and in 5 animals/sex/group at the end of the study.


Sacrifice and pathology
GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS

All animals surviving to the end of the observation period and female no. 46 which was killed moribund on post-coitum Day 24 were deeply
anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem
examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Identification marks: not processed Ovaries
Adrenal glands
(Aorta)
Brain - cerebellum, mid-brain, cortex
Caecum
Cervix
Clitoral gland
Colon (
Coagulation gland
Duodenum
Epididymides
Eyes (with optic nerve (if detectable) and Harderian gland)
Female (and male) mammary gland area
Femur including joint
Heart
Ileum
Jejunum
Kidneys
(Lacrimal gland, exorbital)
(Larynx)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Ovaries
(Pancreas)
Peyer's patches [jejunum, ileum] if detectable
Pituitary gland
Preputial gland
Prostate gland
Rectum
(Salivary glands - mandibular, sublingual)
Sciatic nerve
Seminal vesicles
Skeletal muscle
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid including parathyroid if detectable
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions


In addition of 5 animals/sex/group following organs were preserved:
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus
Uterus (including cervix)
Prostate
Seminal vesicles including coagulating glands
Thyroid including parathyroid


Microscopic examination
Test item-related microscopic findings after treatment with 1,3-divinylimidazolidin-2-one were noted in the thyroid glands of males and females and the testes and epididymides of males.
Thyroid gland:
Follicular cell hypertrophy was present at increased incidence and/or severity in males treated at 150 (1 minimal, 4 slight) and 450 (2 minimal,
2 slight, 1 moderate) mg/kg bw/day compared to background severities in control (1 minimal) and 50 (3 minimal) mg/kg bw/day treated males.
In females, follicular cell hypertrophy was present at increased incidence and severity at 450 (2 minimal, 2 slight, 1 moderate) compared to
background incidences and severities in control (none), 50 (1 minimal, 1 slight) and 150 (2 minimal, 1 slight) mg/kg bw/day treated females.
Testes:
Multinucleated giant cells were present in all males treated at 450 mg/kg bw/day (4 minimal, 3
slight, 3 marked)
Germ cell degeneration was present in all males treated at 450 mg/kg bw/day (4 slight, 6 marked).
Germ cell depletion was present in all males treated at 450 mg/kg bw/day (1 slight, 9 marked).
Epididymidis:
Cell debris was present in all males treated at 450 mg/kg bw/day (1 slight, 2 moderate, 7 marked).
Reduced sperm was present in all males treated at 450 mg/kg bw/day (2 slight, 3 moderate, 5
marked).
Other findings:
Hematopoiesis in the spleen was present at slightly increased severity in 450 mg/kg/day treated females

There was one male (number 6) of the control Group 1 that failed to sire (the female which was paired with this male, number 46, was killed in
extremis). There were three females with total litter loss, female 57 treated at 50 mg/kg bw/day, female 67 treated at 150 mg/kg bw/day and female 71 treated at 450 mg/kg bw/day. No abnormalities were seen in the reproductive organs and mammary glands of these animals, which could account
for this.
There were morphological findings in the reproductive organs of the males treated at 450 mg/kg
bw/day which could be attributed to the test item. The spermatogenic staging profiles were abnormal
for these males.
There were no morphological findings in the reproductive organs of the males treated at 50 and 150 mg/kg bw/day which could be attributed to the
test item. The spermatogenic staging profiles were normal for these males.
There were no morphological findings in the reproductive organs of the females which could be attributed to the test item.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, spermatogenesis.
Slides of the testes were made of all males of Groups 1 and 4 and stained with PAS/haematoxylin to assess spermatogenesis.

Litter observations:

Mortality / Viability
The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical signs
At least once daily, detailed clinical observations were made for all animals.
Body weights
Live pups were weighed on Days 1 and 4 of lactation.
Sex
Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

SACRIFICE
Females which delivered: Lactation Days 5-6.
Females with total litter loss (nos. 57, 67, 71) :Within 24 hours of litter loss.
Males: Following completion of the mating period (a minimum of 28 days of dose administration).
Euthanized in extremis (no. 46): When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.
1(Recognizable fetus was examined externally and sexed.)
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on days 5-6 days of lactation.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of
pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.
If possible, defects or cause of death were evaluated.

Statistics:

Statistics
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Reproductive indices:

Mating index (%) Number of females mated x 100 / Number of females paired

Fertility index (%) Number of pregnant females x 100 / Number of females paired

Conception index (%) Number of pregnant females x 100 / Number of females mated

Gestation index (%) Number of females bearing live pups x 100 / Number of pregnant females

Duration of gestation Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live females at first litter check: Number of live female pups at First Litter Check x 100 Number of live pups at First Litter Check

Percentage live males at first litter check: Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage of postnatal loss: Number of dead pups before planned necropsy x 100 / Number of live pups at First Litter Check

Viability index: Number of live pups before planned necropsy x 100 / Number of pups born alive

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Mortality:
One female of the control group (no. 46) was sacrificed moribund on post-coitum Day 24. On this day, dried blood was noted in the cage.
Macroscopic findings in this female included black-brown fluid in the uterus and vagina and beginning autolysis of a fetus in the uterus. These findings likely caused the moribundity of this female. Further one empty implantation site was noted in the uterus. There were no
microscopic findings indicative of the cause of moribundity.
Three females were sacrificed on Day 2 or 3 of lactation because of total litter loss: no. 57 (50 mg/kg), no. 67 (150 mg/kg) and no. 71 (450 mg/kg).

Clinical signs
Towards the end of the study, all males and one female at 450 mg/kg bw/day showed hunched posture. Additionally, piloerection was observed in all males at 450 mg/kg bw/day on a single day in their last treatment week, in three females at 450 mg/kg bw/day during the lactation period for 1, 3 or 5 days, and in one female at 150 mg/kg bw/day on a single day during lactation. These clinical signs
were considered to be related to treatment. Salivation was noted after dosing in both sexes at all dose levels in a dose-related manner. This
salivation was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Incidental findings that were noted included scabs on the cheek, alopecia and rales. These findings
occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance

Body weights
In males at 150 and 450 mg/kg bw/day body weights and body weight gain were lower throughout the treatment period. The differences from control values were generally statistically significantly and showed a dose-related response. Towards the end of the study (Day 15 of the mating period),
mean body weights were 8% and 11% lower than in controls at 150 and 450 mg/kg bw/day, respectively. Females at 450 mg/kg bw/day had lower
body weights and body weight gain during gestation and lactation. The differences from controls were statistically significant, except for mean body weight on post-coitum Day 4 and body weight gain during lactation. In females at 50 and 150 mg/kg bw/day cumulative body weight gain was
statistically significantly lower than in controls at post-coitum days 11 through 20. However, there was no dose-related response and mean body
weights at these dose levels were similar to control values. Therefore, the differences in body weight gain at 50 and 150 mg/kg bw/day were not
attributed to treatment.

Food consumption:
In males at 450 mg/kg bw/day food consumption (before and after allowance for body weight) was lower than that in controls during the premating
period. To a lesser extent, food consumption before allowance for body weight in these males was also slightly lower during the mating period.
In females at 450 mg/kg bw/day food consumption before and after allowance for body weight were lower than in controls during the second week of the premating period (Days 8-15). Food consumption of these females was also lower during gestation and lactation but only before allowance for
body weight (differences from controls were statistically significant at post-coitum Days 14-17 and 17-20).

Clinical biochemistry details
The following changes in clinical biochemistry parameters distinguished animals treated at 450 mg/kg bw/day from control animals:
Lower total protein in both sexes (statistically significant in males only). Albumin was also slightly lower but the differences from control were not
statistically significant.
Higher total bilirubin in males (statistically significant).
Higher creatinine in both sexes (statistically significant in males only).
Higher potassium in males (statistically significant).
Lower sodium in females (statistically significant).
Lower inorganic phosphate in females (not statistically significant).
The other routine clinical biochemistry parameters showed no treatment-related or toxicologically relevant changes.
Statistically significant differences noted for ALP (lower) and glucose (higher) at 150 mg/kg bw/day in males were not attributed to treatment
because there was no dose-related response. Further it was noted that the mean plasma levels of ALAT (males) and bile acids (both sexes) at
450 mg/kg bw/day were higher. The differences from controls were not statistically significant. The higher mean ALAT value was particularly due
to a high value in one animal (no. 32). The bile acid values in individual animals showed wide variation with low values in a male (no. 3) and a female (no. 48) of the control group.

Thyroid hormones:
There was a treatment-related decrease in total T4 levels in both sexes. In males total T4 levels were statistically significantly lower at all dose levels, most markedly at 450 mg/kg bw/day (at 50 and 150 mg/kg bw/day total T4 levels were reduced to the same extent, thus without a clear dose relationship). The total T4 levels in treated males at all dose levels were below the historical control range1 (concurrent controls were at the lower end of this range). In females total T4 levels were decreased at 150 and 450 mg/kg bw/day (as values were below the lower limit of quantification it was not
possible to assess whether the differences from the control group were statistically significant and/or doserelated).
TSH was also affected though less clearly than total T4. Differences from the control group were not statistically significant. TSH values in all females treated at 450 mg/kg bw/day were higher compared to concurrent and historical controls1. TSH was also higher in a single female (no. 51) at
50 mg/kg bw/day and in 2/5 females (nos. 61 and 62) at 150 mg/kg bw/day. In males TSH was higher than controls (concurrent and historical) in
2/5 animals (nos. 32 and 34) at 450 mg/kg bw/day.
Total T3 levels did not indicate an effect of treatment on this hormone. The values were in the historical control range1, except for those in a few
females (one of the control group, one at 150 mg/kg bw/day and one at 450 mg/kg bw/day) which were below the lower limit of quantification (LLOQ).
Organ weights:
Mean terminal body weights were statistically significantly lower at 150 mg/kg bw/day in males (relative difference from control value: 8%) and at 450 mg/kg bw/day in both sexes (relative differences from control: 10%).
Males at 450 mg/kg bw/day had treatment-related and toxicologically relevant changes in the weights (absolute and relative to body weight) of the
testes (mean relative weight 14% lower than control value) and thyroid (mean relative weight 75% higher than control value).
Other statistically significant differences noted in males or females were considered not to be related to treatment because they showed no dose-
related response (higher relative thyroid weight at 50 mg/kg bw/day in males; higher relative prostate weight at 150 mg/kg bw/day; lower weight of
the ovaries at 50 and 450 mg/kg bw/day) or were secondary to the treatment-related decrease in body weight at 450 mg/kg bw/day (lower absolute weights of the epididymides and seminal vesicles in males; lower absolute weight of the heart in females).
The statistically significantly lower absolute thymus weight in females at 450 mg/kg bw/day was neither accompanied by a statistically significant
decrease in relative thymus weight nor by microscopic changes. Therefore, this finding was considered not to be toxicologically relevant. Further it
was noted that the relative weights of the adrenals and spleen were higher (not statistically significantly) at 450 mg/kg bw/day in females. These
differences in organ to body weight ratios were considered to be secondary to the lower terminal body weight of females at 450 mg/kg bw/day.
Moreover, the higher mean weight of the adrenals was particularly due to the high value in one female (no. 77).
Other organ weights and organ to body weight ratios among the dose groups were similar to those of control animals.

Histopathology
Thyroid gland:
Follicular cell hypertrophy was present at increased incidence and/or severity in males treated at 150 (1 minimal, 4 slight) and 450 (2 minimal, 2 slight, 1 moderate) mg/kg bw/day compared to background severities in control (1 minimal) and 50 (3 minimal) mg/kg bw/day treated males.
In females, follicular cell hypertrophy was present at increased incidence and severity at 450 (2 minimal, 2 slight, 1 moderate) compared to
background incidences and severities in control (none), 50 (1 minimal, 1 slight) and 150 (2 minimal, 1 slight) mg/kg bw/day treated females.
Testes:
Multinucleated giant cells were present in all males treated at 450 mg/kg bw/day (4 minimal, 3 slight, 3 marked)
Germ cell degeneration was present in all males treated at 450 mg/kg bw/day (4 slight, 6 marked).
Germ cell depletion was present in all males treated at 450 mg/kg bw/day (1 slight, 9 marked).
Epididymidis:
Cell debris was present in all males treated at 450 mg/kg bw/day (1 slight, 2 moderate, 7 marked).
Reduced sperm was present in all males treated at 450 mg/kg bw/day (2 slight, 3 moderate, 5 marked).
Other findings:
Hematopoiesis in the spleen was present at slightly increased severity in 450 mg/kg/day treated females.
Spleen:
In this study the control females had a low severity of hematopoiesis compared to what is normally found in this
type of studies.
There were no other test item-related histologic changes.

There was one male (number 6) of the control Group 1 that failed to sire (the female which was paired with this male, number 46, was killed in
extremis). There were three females with total litter loss, female 57 treated at 50 mg/kg bw/day, female 67 treated at 150 mg/kg bw/day and female
71 treated at 450 mg/kg bw/day. No abnormalities were seen in the reproductive organs and mammary glands of these animals, which could account for this.There were morphological findings in the reproductive organs of the males treated at 450 mg/kg bw/day which could be attributed to the test item. The spermatogenic staging profiles were abnormal for these males.
There were no morphological findings in the reproductive organs of the males treated at 50 and 150 mg/kg bw/day which could be attributed to the
test item. The spermatogenic staging profiles were normal for these males.
There were no morphological findings in the reproductive organs of the females which could be attributed to the test item.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: testes and epididymides

Results: F1 generation

Details on results (F1)

Gestation
The gestation index and duration of gestation were unaffected by treatment up to 450 mg/kg bw/day.
Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no
signs of abortion or premature birth, except for the cage debris of one control female (no. 46) in which dried blood was seen on post-coitum Day
24 (this female was subsequently sacrificed). No deficiencies in maternal care were observed.
Early postnatal pup development
The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
Mortality
At the first litter check, two pups of the control group, one pup at 150 mg/kg bw/day and two pups at 450 mg/kg bw/day were found dead.
During lactation, 12 pups of the control group, 19 pups at 50 mg/kg bw/day, 3 pups at 150 mg/kg bw/day and 15 pups at 450 mg/kg bw/day were missing or found dead. At 150 mg/kg bw/day, the totalnumber of pups lost postnatally (lower) and viability index (higher) differed statistically
significantly from control values. These differences were not toxicologically relevant as they reflected good survival at 150 mg/kg bw/day. Pups
missing were most likely cannibalised. The postnatal loss was relatively high in the control group and at 50 and 450 mg/kg bw/day. In the absence of a dose-related response this was considered not to be related to treatment.
Clinical signs
Clinical signs in pups that went missing or were found dead included absence of milk in the stomach, insufficient milk in the stomach, emaciation,
laboured respiration and purple discolored. Incidental clinical signs among surviving pups included scabs on the snout or head, a bent tail, blue spot on the head, absence of milk in the stomach and insufficient milk in the stomach. The nature and incidence of these findings remained within the
range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Body weights
Body weights of pups were not adversely affected by treatment.
At 50 and 450 mg/kg bw/day mean body weights of male and female pups were slightly lower on Days 1 and 4 of lactation. The differences from
control values were not statistically significant and showed no dose-related response (values at 150 mg/kg bw/day were similar to control values).
Therefore, this finding was considered not to be toxicologically relevant.
Macroscopy
Findings in pups that were found dead included advanced or beginning autolysis, a wound in the abdominal region, partial cannibalism and absence of milk in the stomach. Macrosocopic findings among surviving pups were limited to a bent tail in one pup at 50 mg/kg bw/day. The nature and
incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no
toxicological relevance.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Treatment-related toxic effects in the testes and epididymides were observed at 450 mg/kg bw/day. The testicular changes consisted of a decrease in testes weight, a flaccid appearance at macroscopic examination and histopathological changes. The latter changes occurred in all males treated at 450 mg/kg bw/day and were characterized by germ cell degeneration, germ cell depletion, the presence of multinucleated giant cells and abnormal spermatogenic staging profiles. Changes in the epididymides consisted of the presence of cell debris and a marked reduction of the amount of sperm in this organ. Sperm is produced in the testes and thereafter

it is transported to the epididymides were it becomes fully matured while being transported from the head to the tail of the

epididymides. This transport takes time and is the reason that, despite the degeneration/depletion of germ cells, there was no decrease in the number of pregnant females at 450 mg/kg bw/day (after 14 days of treatment during the premating period, there was still viable sperm left in the epididymides). At the time that the males were sacrificed (after four weeks of treatment), there was no sperm left in the first three quarters of the epididymides and only some sperm mixed with cell debris was present in the last part (the tail). This indicates the test item has no direct effect on the sperm and its quality, but rather has an effect on the germ cells in the testes, or on the supporting Sertoli cells.

No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, and histopathological examination of female reproductive organs)

Applicant's summary and conclusion

Executive summary:

In conclusion, treatment with 1,3-divinylimidazolidin-2-one by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 450 mg/kg revealed parental toxicity at all dose levels. Adverse parental effects at 450 mg/kg bw/day included clinical signs, reduced body weight and food consumption, signs of anaemia, changes in clinical biochemistry parameters and changes in thyroid

hormones, thyroid weight and thyroid morphology. Effects on body weight and thyroid (hormones and morphology) were also noted at 150 mg/kg bw/day. The only change at 50 mg/kg bw/day was a decrease in the serum level of thyroid hormone T4 (total T4) in males. Reproduction toxicity was noted in males at 450 mg/kg bw/day and consisted of effects on the testes (reduced weight,

histopathological changes) and epididymides (histopathological changes). No developmental toxicity was observed for treatment up to 450 mg/kg bw/day.

Based on the results of this study, the following No Observed Adverse Effect Levels (NOAEL) were

derived:

Reproduction NOAEL: 150 mg/kg bw/day

Developmental NOAEL: at least 450 mg/kg bw/day