Perhydroimidazo[4,5-d]imidazole-2,5-dione

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

OECD 422 - Combined Repeated Dose Toxicity Study with the Reproduction/Development Toxicity Screening Test

Type of information:

experimental study

Remarks:

N,N’-(isobutylidene)diurea is the structural analogue.

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 - Combined Repeated Dose Toxicity Study with the Reproduction/Development Toxicity Screening Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-(isobutylidene)diurea

EC Number:

228-055-8

EC Name:

N,N''-(isobutylidene)diurea

Cas Number:

6104-30-9

Molecular formula:

C6H14N4O2

IUPAC Name:

[1-(carbamoylamino)-2-methylpropyl]urea

Specific details on test material used for the study:

Batch number: 1/1/98.
Purity: 90.1% (no correction factor was used for the preparation of the dosage forms).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl CD® (SD) IGS BR strain, Caesarian Obtained, Barrier Sustained- Virus Antibody Free, (COBS-VAF®). Breeder: Charles River Deutschland GmbH.

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Strain and sanitary status: Crl CD® (SD) IGS BR strain, Caesarian Obtained, Barrier Sustained- Virus Antibody Free, (COBS-VAF®). Breeder: Charles River Deutschland GmbH.
Identification: Parent animals were individually identified by an ear tattoo (unique CIT identity number). The pups were individually identified by subcutaneous injection of Indian ink in the paws.
Test group and doses: Animals grouped per dose (0, 100, 300, 1000 mg/kg/day).
Number of animals: 10males/10females per dose group.
Acclimation period: 6-day acclimation period, a larger number of animals than necessary were acclimated to permit selection and/or replacement of individuals.
Allocation to study: 40 males/40 females selected according to body weight and/or clinical condition and randomly allocated by sex to the groups.
Age: 8 week old males, 10 week old females.
Weight: Males mean 276g (range: 259g to 306g), Females mean 228g (range: 213g to 248g).

ENVIRONMENTAL CONDITIONS:
Animals were housed in barriered rodent unit; animal room was disinfected before arrival of animals and cleaned regularly thereafter.
Temperature : 21± 2°C.
Relative humidity : 50 ± 20%.
Light/Dark cycle : 12h/12h (07:00 - 19:00).
Ventilation : ~12 cycles/hour of filtered, non-recycled air.
Housing: Housed individually in suspended wire-mesh cages (43.0 x 21.5x18.0 cm). A metal tray containing autoclaved sawdust (SICSA, Alfortville, France), was placed under wire-mesh cages. Four days before the lactation period the females were housed individually in polycarbonate cages (43.0 x 21.5 20.0 cm) containing autoclaved sawdust with wood shavings (SICSA, Alfortville, France). The cages were placed in numerical order on the racks.
Food: Ad libitum (A04 C pelleted diet - batch no's. 00331 and 00516 (UAR,Villemoisson, Epinay-sur-Orge, France) distributed weekly.
Water: Ad libitum (filtered tap water).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

The vehicle was 0.5% aqueous CMC solution.

Details on exposure:

The dosage forms were administered by gavage using a plastic syringe fitted with a metal gavage tube. Quantity of the dosage form administered to each animal was adjusted according to the most recently recoded body weight. A constant dosage-volume of 10mL/kg/day was used. Dosage forms were stirred continuously throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Remarks:

Analyses were carried out via HPLC with external calibration.

Details on analytical verification of doses or concentrations:

Analyses of the test substance preparations: concentration and homogeneity. Three samples of each control and test substance dosage form (top, middle and bottom of the flasks) prepared for use during the first week and the last week of treatment were taken. Analyses were carried out via HPLC with external calibration.

Sample preparation for analysis: sample was transferred in a flask with doubly distilled water. The sample-solution was treated for 60min in an ultrasonic bath. The mixture was filled up with eluant to the final volume. Aliquots of these dilutions were used for HPLC-analysis.

Equipment:
Column: LiChrosorb NH2 5um [250X4]mm.
Eluant: Acetonitrile: doubly distilled water.H3P04[95/4.9/0.1].
Flow rate: 1.5mL/min.
Detection: UV, 190nm.

Low standard deviation in homogeneity analyses concluded that the test substance was distributed homogeneously in 0.5% CMC in doubly distilled water. Results also demonstrated correctness of the concentrations of the test substance in 0.5% CMC in doubly distilled water.

Details on mating procedure:

Females were mated with males from the same dose-level group: one female was placed with one male, in the latter's cage, during the night. Confirmation of mating was made in the morning by checking the presence of vaginal plug or of sperm in a vaginal lavage. The day of confirmed mating was designated day zero (0) post-coitum.

Each female was placed with the same until mating occurred or 14 days had elapsed. The pre-coital time was calculated for each female.

Duration of treatment / exposure:

In males: 15 days before mating, during the mating and post-mating periods until sacrifice (at least 4 weeks in total).
In females: 15 days before mating, during the mating period, pregnancy and lactation period, in day 4 post-partum inclusive (or until sacrifice, for un-mated females).

Frequency of treatment:

Once a day, at approximately the same time.

Duration of test:

At least four (4) weeks in total.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Animal numbers
Males: W25991 to W26000.
Females: W26071 to W26080.

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Animal numbers
Males: W26001 to W26010.
Females: W26081 to W26090.

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Animal numbers
Males: W26011 to W26020.
Females: W26091 to W26100.

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Animal numbers
Males: W26021 to W26030.
Females: W26101 to W26110.

No. of animals per sex per dose:

Ten (10).

Control animals:

yes, concurrent vehicle

Details on study design:

Three groups of 10 males and 10 females received the test substance by oral gavage once a day at 100, 300 or 1,000 mg/kg/day, as follows:
- Throughout pre-mating period (15 days), during the mating and post-mating periods until final sacrifice, in the males (at least 4 weeks in total),
- Throughout pre-mating (15 days) and mating period, during pregnancy and lactation, until day 4 post-partum inclusive (or until sacrifice for un-mated females), in the females .

A group of 10 males and 10 females was given the vehicle (0.5% aqueous CMC) under the same experimental conditions and acted as a control group.

0 mg/kg body weight/day: as the expected no observed adverse effect level.
100-300 mg/kg body weight/day: as the intermediate dose level and eventually as higher no observed adverse effect level.
1,000 mg/kg body weight/day: as the dose level where toxic effects were expected.

Examinations

Maternal examinations:

CLINICAL EXAMINATIONS

MORTALITY: Each animal was checked at least twice a day for mortality and signs of morbidity.
GENERAL CLINICAL OBSERVATIONS: Each animal was observed at least once a day, at approximately the same time for the recording of clinical signs (including evidence of abortion for the females).
DETAILED CLINICAL OBSERVATIONS: All animals of each group were observed in the cage, in the hand and in the standard arena, by observers unaware of the animal's treatment, before the first day of treatment and then once a week thereafter. The animals were randomized in order to ensure "blind" evaluation, except for the examination performed before the first day of treatment. Following parameters were assessed:
- "Touch escape" or ease of removal from cage;
- In the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
- In the standard arena (2-minute recording): grooming, palpebral closure, defecation and urination counts, tremors, twitches, convulsions, gait, arousal (hypo- and hyperactivity), posture, stereotypy, behaviour, breathing, ataxia and hypotonia.
REACTIVITY TO MANIPULATION OR TO DIFFERENT STIMULI: In five (5) males and five (5) females per dose group, the examinations below were conducted shortly before terminal sacrifice. "Blind" evaluation was carried out.
- Touch response, forelimb grip strength, pupil reflex, visual stimulus, auditory startle reflex, tail pinch response.
MOTOR ACTIVITY: Measured in five (5) males and five (5) females per dose group, the examinations below were conducted shortly before terminal sacrifice, using an automated infra-red sensor equipment recording individual animal activity over a 30-minute period (except for the females: 10 minutes).
BODY WEIGHT: Males: Each animal was recorded on the first day of treatment (day 1), then once a week until sacrifice.
Females: Each animal was recorded on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14, 20 post-coitum and days 1 and 4 post-partum.
FOOD CONSUMPTION: Quantity of food consumed by each animal was recorded once a week, from the first day of each of the pre-mating, gestation and lactation periods. During the mating period, the food consumption was noted for neither males nor females.

LABORATORY INVESTIGATIONS

BLOOD AND URINE COLLECTION: Blood samples taken from the orbital sinus of the animals under isoflurane anaesthesia and collected into tubes containing the appropriate anticoagulant. Note: prior to blood sampling and during urine collection; animals were deprived of food. For urine collection, the animals were individually placed in metabolism cages for an overnight period of at least 14 hours . The urine was collected in the presence of thymol crystals.
HAEMATOLOGY: Were determined in five (5) males and five (5) females per dose group, at the end of the pre-mating period.
BLOOD BIOCHEMISTRY: Were determined in five (5) males and five (5) females per dose group, at the end of the pre-mating period.
URINALYSIS: Were determined in five (5) males and five (5) females per dose group, at the end of the pre-mating period.

NECROPSY

MACROSCOPIC: A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.
MICROSCOPIC: All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with haematoxylin-eosin (except testes and epididymides which were stained with haematoxylin/PAS). Performed on:
- Five (5) males and five (5) females of the control and high-dose groups (groups 1 and 4) killed at the end of the treatment period and for the female W26092 found dead,
- All macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) killed on completion of the treatment period,
- All kidneys of all male animals,
- Genital organs (testes, epididymides, prostate, seminal vesicles, ovaries, uterus) of all male and female animals of any group.

Ovaries and uterine content:

NECROPSY

MICROSCOPIC EVALUATION: Histopathological examination of the ovaries aimed at detecting qualitative depletion of the primordial follicle population. In the parent females, the corpora lutea and implantation sites were recorded. In apparently non-pregnant or un-mated females, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique.

Fetal examinations:

LITTER SIZE: All pups derived from the F0 parents were examined as soon as possible on the day of birth to determine the total number of litter size and numbers of pups of each sex. The litters were observed daily in order to note the number of live, dead and cannibalised pups and runts. Any gross abnormality was noted.

BODY WEIGHT: The weight of each pup was recorded on days 1 and 4 post-partum.

CLINICAL SIGNS: The pups were observed daily for clinical signs or abnormal behaviour.

NECROPSY MACROSCOPIC: Dead pups and those killed on day 5 post-partum were carefully examined externally for gross abnormalities.

Statistics:

Data are expressed as group mean values t-standard deviation or as proportions. Whenever necessary, the experimental unit of comparison was the litter.
Mean quantitative values are compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Proportions are compared by Fisher exact probability test.

Indices:

OBSERVATION OF PUPS AT BIRTH

There were no stillborn pups or runts. The number of live born pups was similar in the control and the 100 and 300 mg /kg/day groups. In the 1000 mg/kg/day group, the number of pups per litter was slightly lower (12.9 versus 14.7 per female). As mentioned above, this was principally due to a single female with a low litter size, and thus, was considered to be fortuitous. There were no gross external abnormalities in the control and the treated groups.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One female in the 300mg/kg/day group died on day 14 of the pre-mating period after blood sampling.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Females: The body weight change of the females given 100 or 300 mg/kg/day was similar to that of the control group during the pre-mating, pregnancy and lactation periods. In the 1000 mg/kg/day treated group, the body weight change was similar during the pre-mating period, but was slightly lower during pregnancy (day 0 to day 20 of pregnancy:- 10%, not statistically significant) and lactation (day 1 to day 4 post-partum:-38%, not statistically significant). Mean body weights on day 0 of pregnancy and day 4 post-partum statistically differed from that of the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females: The food consumption of all the treated females was similar to that of the control females, during the pre-mating and post-mating periods except for females of the 1000 mg/kg/day group in which a slight reduction in food consumption was recorded during the pregnancy (day 0 to day 20 of pregnancy:-6%, not statistically significant).

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Notable changes in biochemistry parameters were noted in the treated males and females across all dose ranges, however they were not considered to be related to the treatment with the test substance since they were slight, not dose-related and/or statistically significant. Furthermore, the values were within the range of the labs historical control data and as such observed effects were considered to be of fortuitous occurrence.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

NECROPSY
MICROSCOPIC EXAMINATION: Kidneys; there appears to be a dose-related higher severity of acidophilic globules in the cortical tubular epithelium of the kidneys of the treated male rates, given 300 and 1000mg/kg/day. No tubular degeneration/necrosis was observed in the kidneys of the males of any group, the presence of acidophilic globules was considered to be of minor toxicological importance and most probably due to the accumulation of the sex-linked alpha 2µ-globulin in the cortical tubular cells of the male rats. This is a sex-and species-related effect, as such no accumulation occurs in female rats or in humans.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

1000mg/kg/day of the test substance revealed slight signs of maternal (but not paternal) toxicity, confined to:
- a slightly lower food consumption in the females during gestation (-6%, compared to controls, day 0-day 20 of pregnancy),
- a slightly lower body weight gain in the females during gestation (-10%, compared to controls, day 0-day 20 of pregnancy) and lactation (-38%, compared to controls, day 1-day 4 post-partum).

No signs of substance-induced paternal or maternal toxicity occurred at the mid and low ~dose-levels (100 or 300 mg/kg/day). At all three dose-levels, there was a slightly to moderately higher incidence and/or severity of the presence of acidophilic globules in the kidneys of the males, at microscopic examination. Since no tubular degeneration/necrosis was observed in the kidneys of the animals of any group, this finding was considered to be of minor toxicological importance and was not considered to be an adverse effect. In addition, this finding, which is known to be sex- and species-specific, has no relevance for humans in terms of risk assessment.

No substance-related effects were observed at any dose-level (including and up to 1000 mg/kg/day) on the male and female reproductive performance. In particular, there were no effects on the mating and fertility parameters and the development of the pups after birth was not affected. Histopathological investigations confirmed that there were no substance-induced changes in the reproductive organs.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

not specified

Other effects:

not specified

Details on maternal toxic effects:

No developmental maternal toxic effects were observed.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

dead fetuses

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

number of abortions

other: see 'remark'

Remarks on result:

other: Reproductive and developmental toxicity.

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

mortality

other: See 'Remark'

Remarks on result:

other: Parental toxicity.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The weights of pups were similar in the control and the treated groups on day 1 and day 4 post-partum (being slightly higher in the high-dose group , as expected for lower-sized litters).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

The number of liveborn pups was similar in the control and the 100 and 300 mg /kg/day groups. In the 1000 mg/kg/day group, the number of pups per litter was slightly lower (12.9 versus 14.7 per female). As mentioned above, this was principally due to a single female with a low litter size, and thus, was considered to be fortuitous.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See 'Remarks'

Remarks on result:

other: Reproductive and developmental.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

An OECD TG 422 (2003) study was conducted to assess the repeat-dose and the potential reproduction/developmental toxicity of isobutylidene-di-urea, a suitable analogue for the target substance glycoluril (further information documented in the read-across justification documentation). Three groups of ten (10) males and ten (10) females received the test substance by oral gavage once a day at 100, 300 or 1,000 mg/kg/day. A group of ten (10) males and ten (10) females was given the vehicle (0.5% aqueous CMC) under the same experimental conditions and acted as a control group.

Mortality and clinical signs were checked daily. Blood and urine samples were collected at the end of the pre-mating period from five (5) males and five (5) females per dose group for blood biochemistry and urinalysis investigations. During the lactation period, pups were examined daily for survival, external abnormalities and clinical signs. The body weight was also recorded on days one (1) and four (4) post-partum.

At the final sacrifice of the parents, specified organs were weighed and a complete macroscopic necropsy examination was performed. A microscopic examination was performed on a range of sample tissues and organs for five animals of the control and highest dosage-group and in animals of the low and intermediate groups which presented macroscopic lesions. Genital organs were examined in all male and female animals. Kidneys were examined in all male animals.

Animals who received the highest dose (1,000mg/kg/day) were observed to have the following substance-related effects:
- Slightly lower body weight gain in the females during gestation (-10%, compared to controls, day 0-day 20 of pregnancy) and lactation (-38%, compared to controls, day 1-day 4 post-partum),
- Slightly lower food consumption in the females during gestation (-6%, compared to controls, day 0-day 20 of pregnancy),
- No substance-related effects on the male and female reproductive performance and no indication for substance-induced developmental toxicity.

Animals who were at the low (100mg/kg/day) and mid-dose (300mg/kg/day) ranges were observed to have no substance-related effects on male and female parental rats and no indication for substance-induced developmental toxicity.

In conclusion, under the conditions of this study, the oral administration of isobutylidene-di-urea elicited slight maternal toxicity at 1000mg/kg/day in the females. Maternal toxicity was substantiated in this group by reduced food consumption and body weight gain. These findings were not observed in the males of this group. No signs of substance-induced maternal or paternal toxicity occurred at the low or mid dose-levels (100 or 300mg/kg/day; respectively). There were no substance-induced effects on the male and female reproductive performance, and on the progeny of the parental rats up to and including the highest dose-level (1,000mg/kg/day).

Based on the results discussed above, the no observed adverse effect level (NOAEL) for parental toxicity of isobutlylidene-di-urea is 300mg/kg/day, while the NOAEL for the toxic effect on reproductive performance and on developmental toxicity is 1,000mg/kg/day. As such, the substance does not meet the classification criteria for reproductive effects and developmental effects under Regulation No.1272/2008.

Executive summary:

An OECD TG 422 (2003) study was conducted to assess the repeat-dose and the potential reproduction/developmental toxicity of isobutylidene-di-urea. Three groups of ten (10) males and ten (10) females received the test substance by oral gavage once a day at 100, 300 or 1,000 mg/kg/day. A group of ten (10) males and ten (10) females was given the vehicle (0.5% aqueous CMC) under the same experimental conditions and acted as a control group.

 

No signs of substance-induced maternal or paternal toxicity occurred at the mid or low dose-levels (100 or 300 mg/kg/day). There were no substance-induced effects on the progeny of the parental rats up to and including the highest dose-level (1000 mg/kg/day).

 

Based on these results , the no observed adverse effect level (NOAEL) for developmental toxicity is 1000 mg/kg/day. As such the substance is not classified under Regulation (EC) No. 1272/2008.