1,3-divinylimidazolidin-2-one

Endpoint summary

Description of key information

NOAEL: 50 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on oral exposure:

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

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

Remarks:

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

No. of animals per sex per dose:

10 animals/sex/group;
5 animals/sex/group were selected for functional observations, locomotor activity, clinical pathology, organ weights and histopathology

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 14-day range-finding study in rats in which dose levels of 100, 300 and 1000 mg/kg bw/day
were tested.

Observations and examinations performed and frequency:

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 this was also performed outside the home cage in a
standard arena.

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.

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:
Haematology
White blood cells
Differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils)
Red blood cells
Reticulocytes
Red blood cell distribution width
Haemoglobin
Haematocrit
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
Aspartate aminotransferase
Alkaline phosphatase
Total Protein
Albumin
Total Bilirubin
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate
Bile acids

Thyroid hormone analyses:
Thyroid stimulating hormone (TSH) μIU/mL
total triiodothyronine (T3) ng/dL
total thyroxine (T4) μg/dL


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 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.
Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4%
formaldehyde solution.

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)
(Esophagus)
(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

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett-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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

hunched posture, mostly in males and piloerection in males and females

Mortality:

mortality observed, treatment-related

Description (incidence):

hunched posture, mostly in males and piloerection in males and females

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced body weight at 150 mg/kg in males and at 450 mg/kg in both sexes

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

females at 450 mg/kg lower number of red blood cells, lower haematocrit and lower haemoglobin concentration. higher values of reticulocytes and red blood cell distribution width

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

testes; thyroid glands

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

testes; epididymides; thyroid glands

Histopathological findings: neoplastic:

not examined

Details on results:

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).

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 endof 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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: thyroid glands

Critical effects observed:

not specified

Executive summary:

There was an increased incidence and severity of follicular cell hypertrophy at 150 mg/kg bw/day in males and at 450 mg/kg bw/day in both sexes, an increased weight of the thyroid (absolute and relative to body weight) at 450 mg/kg bw/day in males, and a higher serum level of TSH at 450 mg/kg bw/day in both sexes observed. TSH was also higher in one female at 50 mg/kg bw/day and two females at 150 mg/kg bw/day. Lower serum levels of the thyroid hormone T4 (total T4) were observed at all dose levels in males and at 150 and 450 mg/kg bw/day in females. In males a clear dose relationship was absent. Further, the lower value for total T4 in males at 50 mg/kg bw/day was slight and not accompanied by histopathological changes in the thyroid. Therefore, this difference was considered not to be toxicologically relevant.

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 150 and 450 mg/kg bw/day. 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. Treatment-related toxic effects in the testes and epididymides were observed in males at 450 mg/kg bw/day and consisted of reduced testes weight and histopathological changes in the testes and epididymides. No developmental toxicity was observed for treatment up to 450 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There was an increased incidence and severity of follicular cell hypertrophy at 150 mg/kg bw/day in males and at 450 mg/kg bw/day in both sexes, an increased weight of the thyroid (absolute and relative to body weight) at 450 mg/kg bw/day in males, and a higher serum level of TSH at 450 mg/kg bw/day in both sexes. TSH was also higher in one female at 50 mg/kg bw/day and two females at 150 mg/kg bw/day. Lower serum levels of the thyroid hormone T4 (total T4) were observed at all dose levels in males and at 150 and 450 mg/kg bw/day in females. In males a clear dose relationship was absent. Further, the lower value for total T4 in males at 50 mg/kg bw/day was slight and not accompanied by histopathological changes in the thyroid. Therefore, this difference was considered not to be toxicologically relevant.

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 at150 and 450 mg/kg bw/day. 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. Treatment-related toxic effects in the testes and epididymides were observed in males at 450 mg/kg bw/day and consisted of reduced testes weight and histopathological changes in the testes and epididymides. No developmental toxicity was observed for treatment up to 450 mg/kg bw/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study available

Repeated dose toxicity: via oral route - systemic effects (target organ) glandular: thyroids

Justification for classification or non-classification

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified with STOT RE 2, H373 (target organ: thyroid) under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.