1-(2-hydroxyethyl)imidazolidin-2-one

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-(2-hydroxyethyl)imidazolidin-2-one

EC Number:

223-032-9

EC Name:

1-(2-hydroxyethyl)imidazolidin-2-one

Cas Number:

3699-54-5

Molecular formula:

C5H10N2O2

IUPAC Name:

1-(2-hydroxyethyl)imidazolidin-2-one

Details on test material:

- Name of test material:Hydroxyethyl Ethylene Urea (HEEU)
- Physical state: liquid
- Composition of test material, percentage of components: approximately 75% active ingredient (a.i.) in water.
- Lot/batch No.: DLT 33:99

Test animals

Species:

rat

Strain:

other: Crl:CD®BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Kingston Facility, Stone Ridge, NY)
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: mean 239.7-384.2 (female/male)
- Fasting period before study: no
- Housing: individually housed in stainless steel cages (34 cm x 18 cm x 18 cm) with wire-mesh fronts and bottoms, suspended over pans containing absorbent liners. After mating. females were housed in polycarbonatc shoe-box cages (53 em x 29 em x 20 em) containing Alpha-Dri® bedding (Shepherd Specialty Papers, Inc.) throughout gestation and lactation.
- Diet (ad libitum): PMI Certified Rodent Diet #5002M (Ralston Purina Co., St. Louis, MO)
- Water (ad libitum): Water purified by reverse osmosis was available via an automatic watering system or water bottles.
- Acclimation period: yes, for 10 days prior to the initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22°C
- Humidity (%): 46 to 52%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of HEEU was used to prepare sufficient quantities of gavage solutions at concentrations appropriate to deliver dose levels of 100, 300, and 1000 mg ai/kg/day at a constant volume of 5 ml/kg. The test substance was weighed in a hood and diluted in reverse osmosis (RO) water. Group 1 (control) received RO water only. Gavage solutions were prepared once a week and stored in the refrigerator. Prior to gavaging the animals each day, an aliquot of each dose was placed in a small beaker and allowed to warm to approximate room temperature.

Details on mating procedure:

Adult female rats were placed individually with an assigned male from the same treatment group, and observed daily until copulation was verified by the presence of a sperm plug in situ, multiple sperm plugs on the cage liner (at least 3), and/or sperm in a vaginal lavage sample. The day that positive evidence of mating was observed was designated as Day 0 of gestation, or GO. Animals were paired for a maximum period of 2 weeks. Females that showed no evidence of copulation were assumed pregnant at the end of the mating period, and housed accordingly. On GO, all females were housed individually in polycarbonate cages (53 cm x 29 cm x 20 cm) containing Alpha-Dri® bedding (Shepherd Specialty Papers, Inc.). From G20 on, females were observed twice daily for signs of delivery and/or offspring.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of HEEU in RO water, when stored refrigerated for 8 days, was determined from the first dose preparation. Homogeneity was determined by analyzing top, middle and bottom samples from the first dose preparation. Samples of dosing solutions prepared on days 28 and 56 were analyzed for active ingredient content to determine proximity to target concentrations. Analyses were conducted by a method which was previously verified and approved.

Duration of treatment / exposure:

Exposure of parental animals (12/sex/dose) began when the animals were approximately eleven weeks of age. Parental animals were mated after two weeks of exposure. Treatment continued throughout gestation, lactation, and until terminal necropsy.

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 rats/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The limit dose of 1000 mg a.i./kg/day was selected based on a gavage range-finding study which induced minimal toxicity in the parameters measured. The low and middle doses were approximately logarithmically spaced from the high dose.
- Rationale for animal assignment: the treatment groups using a computerized randomization procedure based on body weight. Following randomization, group mean body weights were visually examined for homogeneity. Rats not assigned to a group were used for health monitoring or returned to the Laboratory Animal Services Unit.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observations for morbidity, mortality or obvious signs of toxicity were performed daily throughout the study.Cage liners were examined daily for gross abnormalities in the feces or urine. During delivery, any signs of difficult or prolonged parturition were noted.
Detailed Clinical Observations (DCO) were performed weekly outside the home cage, in a standard arena, beginning one week prior to the start of dosing. The DCO noted changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern), if present. Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) were recorded if present.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly outside the home cage, in a standard arena, beginning one week prior to the start of dosing. The DCO noted changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern), if present. Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) were recorded if present.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for male animals throughout the study. Females were weighted weekly until cohabitation, on Gestation Days (G) 0,3,6,9, 12, 15, 18 and 20, and on Postnatal Days (PND) 0 and 4.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly during premating for male and female animals until cohabitation. Feed consumption was not measured for either sex during cohabitation due to the inability to ascribe consumption to individual animals. Weekly feed consumption measurements resumed for male animals after cohabitation and continued throughout the study. Maternal feed consumption was measured from GO-7, 7-14, and 14-21, and from PND 0-4.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
Hematology and clinical chemistry measurements were performed on all animals at terminal necropsy. Animals were fasted overnight and blood samples were collected just prior to the terminal necropsy. All samples were collected from the abdominal aorta of rats that had been anesthetized with sodium pentobarbital (Nembutal®, 50 mg/ml, Abbott Laboratories, North Chicago, IL) administered intraperitoneally at approximately 0.1 mI/100 g body weight to effect. Blood samples were collected in an order that rotated through treatment groups (i.e., one animal from each treatment group was bled before a second animal from the same group). Once blood was collected, animals were exsanguinated via the abdominal aorta. Necropsy and blood sample collection were performed over two consecutive days (approximately 6 animals/sex/group were necropsied each day).

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Detailed clinical observations (DCO) were performed weekly on all rats beginning one week prior to the start of dosing. DCOs were conducted on the same day each week, split across two days by sex. Each DCO evaluation assessed an animal's behavior in its home cage and an open arena. The animal was observed in the open arena for one minute. Following the 1-minute observation period, the pupillary response was assessed by shining a penlight in each eye. The DCOs were performed by the same individual (primary observer) throughout the study. A different individual (recorder) was present to record the observations.
- A Functional Observational Battery (FOB) was performed on all surviving rats during the 5th week (males) or 7th week (females) of dosing. Each FOB evaluation assessed an animal's behavior in its horne cage and an open arena.
- Motor activity assessment was performed after all FOBs in a given day's replicate were completed. Each rat was placed individually in a stainless steel cage (wire mesh front and bottom; 41 cm x 24 cm x 18 cm) with a passive infrared motion sensor mounted to the cage front.

Oestrous cyclicity (parental animals):

Not specified

Sperm parameters (parental animals):

Not specified

Litter observations:

Postnatal day 0 (PND0) was designated as the day when delivery was complete. The day that delivery began, if different than PND0, was also noted. On PND0, all offspring were examined, and their status (alive, dead, stillborn or uncertain), weight and sex were determined. A non-viable offspring was classified as stillborn if its lungs did not float in water. Offspring were classified as uncertain if status and/or sex could not be determined. On PND4, offspring were individually weighed, sexed and examined for external structural abnormalities and clinical signs of ill health. A record was maintained for all offspring that died during the lactation period. Cageside observations for signs of mortality, morbidity or obvious indications of a toxic effect were performed daily throughout the lactation period.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
A complete gross examination was performed on every parental animal. Adult animals were euthanized by exsanguination via the abdominal aorta under sodium pentobarbital anesthesia (Nembutal®, 50 mg/ml, Abbott Laboratories) administered intraperitoneally. All organs, tissues and body cavities were examined. The brain, kidneys and liver were sliced (pre- or post-fixation as appropriate) for detection of internal lesions.

ORGAN WEIGHTS: Yes
Organ weights (absolute and relative to body weight) were obtained for the following organs from parental animals during the scheduled necropsy:
Males: testes and epididymides
All Parental Animals: adrenals, brain, heart, kidneys, liver, spleen and thymus

HISTOPATHOLOGY: Yes
Microscopic examination of the organs were performed for 5 (randomly selected) parental animals/sex in the high dose group and control group, and in the Group 3 female humanely sacrificed during the course of this study. All tissues exhibiting gross pathological changes were examined microscopically.

Postmortem examinations (offspring):

On PND4, offspring were individually weighed, sexed and examined for external structural abnormalities and clinical signs of ill health. After all offspring data was collected on PND4, litters were euthanized and discarded without further evaluation.

Reproductive indices:

The following reproductive indices were calculated and analyzed using the 2xN Chisquare Test:
Male Mating Index (%) = (Number of males that mated/Number of males used for mating) x 100
Female Mating Index (%)= (Number of females that mated/Number of females used for mating) x 100
Male Fertility Index (%) = (Number of sires/Number of males mated) x 100
Female Fertility Index (%)= (Number of pregnant females/Number of females mated) x 100
Gestation Index (%) = (Number of females with at least one live pup/Number of pregnant females) x 100

Offspring viability indices:

Viability Index (%) = (Number of pups alive on PND 4/Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
No treatment-related deaths or clinical signs of toxicity were noted in female parental animals during premating, gestation or lactation, in pups during lactation, or in males throughout the study at dose levels up to and including 1000 mg a.i./kg. One female (Group 3, 300 mg/kg) was euthanized for humane reasons during week 7.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related effects on body weight or body weight gain among females during the premating, gestation, or lactation periods, or in males throughout the study. A statistically significant decrease in body weight gain noted in females at 1000 mg a.i./kg during lactation (PNDO-4) was considered incidental.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
There were no treatment-related effects on feed consumption in female parental animals during premating, gestation or lactation, or in males throughout the study at dose levels up to and including 1000 mg a.i./kg.

HAEMATOLOGY
No treatment-related effects on hematology parameters or white blood cell differential counts were observed in male or female animals at any dose level up to and including 1000 mg a.i./kg.

CLINICAL CHEMISTRY
No treatment-related effects on any clinical chemistry parameters were observed in male or female animals at any dose level up to and including 1000 mg a.i./kg. A statistically significant increase in calcium was noted in males at 1000 mg a.i./kg. This increase was not considered treatment-related due to the small magnitude of the change and it was only observed in a single sex.

NEUROBEHAVIOUR
- Detailed Clinical Observations (DCO): No treatment-related systemic or neurological effects were seen in any DCO parameters in either sex at any dose level.
There was a statistically significant change noted in females at 100 mg/kg in reaction to handling during the pretest week. This change was considered incidental since it occurred during the pretest week. Statistically significant increases or decreases were noted in the number of rears in females at 100 and 300 mg a.i./kg during week 5 and at all treatment levels during week 6. Since these changes were in opposite direction, and the group mean values of the treated groups were consistent with previous weeks, the statistical significance were considered incidental and not related to treatment. In addition, there was no dose response evident across treated groups and the variability appeared to be in the control group.
- Functional Observational Battery (FOB): No treatment-related systemic or neurological effects were seen in any FOB parameters in either sex at dose levels up to and including 1000 mg a.i./kg. There were no statistically significant or treatment-related effects in any of the animals' reflexes, responses to stimuli, grip strength, or landing hind foot splay. A statistically significant change in reaction to removal from the cage in females at 1000 mg a.i./kg was not considered treatment-related since the change was well within the range of normal behavior for rats.
- Motor Activity (MA): There were no treatment-related effects in either total time spent in movement or total number of movements in either sex at doses up to and including 1000 mg ai/kg. A statistically significant decrease in the total time spent in movement was noted in males at 1000 mg ai/kg. However, the last 30 minutes of motor activity assessment indicated no difference between the 1000 mg ai/kg group males and controls. In addition, there were no treatment-related alterations in any other parameter associated with the animal's level of arousal, i.e., changes in home-cage behavior, reactivity to handling, open arena activity or number of rears, which would corroborate the biological significance of the decrease in the total time spent in movement in the 1000 mg ai/kg. Therefore, the decrease in total time spent in movement in males at 1000 mg ai/kg was judged not treatment-related.

ORGAN WEIGHTS
There were no treatment-related effects on absolute or relative organ weights in males or females at doses up to and including 1000 mg a.i./kg. A statistically significant increase in the absolute heart weight of males at 300 and 1000 mg a.i./kg was considered incidental and not treatment-related. There was no dose response evident, no changes in relative heart weight were seen, and there were no changes in female heart weights at any dose level. In addition, no microscopic changes in the heart were noted in either sex at the high dose.

GROSS PATHOLOGY
There were no treatment-related gross findings in parental animals of either sex at any dose level.

HISTOPATHOLOGY
There were no treatment-related microscopic changes observed in any male or female rats given 1000 mg a.i./kg/day.

MATING AND FERTILITY
There was no treatment-related effect on any endpoint of mating or fertility in any treatment group, including male and female mating and fertility indices, and time to mating. The male and female mating indices (percent of paired animals that mated) was 100%. The male and female fertility indices (percent of matings that resulted in pregnancy) was 100%. The mean time to mating (days until evidence of mating detected) ranged from 2.2-3.2 days

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general 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 effects were observed at the highest dose tested

2

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects were observed at the highest dose tested

Results: F1 generation

Details on results (F1)

GESTATION AND DELIVERY
There was no treatment-related effect on gestation index (percent of pregnant females that delivered live offspring) which was 100% for all dose levels. There were no treatmentrelated effects on gestation length or number of pups per litter in any treatment group. There were no females that delivered an entirely stillborn litter.

LACTATION
There were no treatment-related effects on offspring viability during lactation in any treatment group. The viability index (percent survival of liveborn offspring from PNDO-4) ranged from 98.2-100%. There were no effects on the mean number of implantation sites or in post-implantation loss at any dose level.There were no treatment-related effects on the ratio of male to female pups in any treatment group. There were no effects on pup body weight at any dose. There were no external abnormalities noted.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

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 effects were observed at the highest dose tested

Overall reproductive toxicity

Reproductive effects observed:

not specified