6-methyl-2-oxoperhydropyrimidin-4-ylurea

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991-07-17 to 1991-08-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted similar or equivalent to the appropriate OECD test guideline with acceptable restrictions. The restriction was that the test animals were treated with the test item up to day 16 post coitum only. The study was compliant with GLP. Since this is a read-across from a structural analogue substance (CAS 6104-30-9), the reliability was set from RL1 to RL2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(2001)

Deviations:

yes

Remarks:

(the animals were treated with the test item only up to day 16 post coitum)

GLP compliance:

yes

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

IUPAC Name:

N,N''-(2-methylpropane-1,1-diyl)diurea

Details on test material:

- Name of test material (as cited in study report): Isobutylidene-di-urea
- Physical state: solide/white
- Storage condition of test material: at room temperature
- Stability under test conditions: was proven for 3 h in vehicle by HPLC

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar rats (Chbb:THOM (SPF))
- Source: Karl Thomae, Biberbach an der Riss, Germany
- Age at study initiation: 65-74 days (sexually mature, virgin)
- Weight at study initiation: mean bw approximately 225.4 g
- Fasting period before study: none
- Housing: individually in type DK III stainless steel wire mesh cages, floor area approximately 800 cm² (supplied by Becker & Co, Castrop-Rauxel, Germany)
- Diet: ground Kliba 343 feed rat/mouse/hamster (Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was freshly prepared as a suspension in 0.5% aqueous CMC solution shortly before administration.

VEHICLE
- 0.5% CMC aqueous CMC solution (Tylose CB 30.000; purified carboxymethyl cellulose supplied by Hoechst AG, Frankfurt/Main, Germany)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance suspensions up to 3 h were carried out during the study. Furthermore, samples of the test substance suspensions were sent to the analytical laboratory of the BASF AG twice during the study period for verification of concentrations. The samples which were sent for the first concentration control analyses toward the beginning of the administration period were also used to verify the homogeneity for the samples of the low and high concentrations (100 mg/kg bw and 1000 mg/kg bw). 6 samples (2 from the top, middle, and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
The test substance analysis was carried out by using HPLC.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1-4 untreated females with 1 untreated fertile male
- Length of cohabitation: from 16.00 h afternoon to 7.30 h the next mroning
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

on gestation days 6-15

Frequency of treatment:

once daily

Duration of test:

On day 20 post coitum all animals were sacrificed.

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 400, and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the outcome of a preliminary range-finding study

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observations for mortality was made twice a day on working days and once a day on Saturdays, Sundays and public holidays; observations for clinical signs were made at least once daily on days 0-20 post coitum; more often when clinical sysmptoms of toxicity were elicited

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 1, 3, 6, 8, 10, 13, 15, 17, and 20 post coitum

FOOD CONSUMPTION: Yes

WATER CONSUMPTION: No
- Time schedule for examinations:

HAEMATOLOGY: Yes
- Time schedule for examinations: on day 16 post coitum
- Parameters checked: white blood cell count (total and differential), red blood cell count, haemoglobine, haematocrite, mean corpuscular volume, mean corpuscular haemoglobine concentration, platelet count, thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for examinations: on day 16 post coitum
- Parameters checked: alanine aminotranfrease (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), serum-gamma-glutamyltransferase (SGGT), electrolytes (Na, K, Cl, inorg. Phosphate, Ca, Mg), urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol

URINALYSIS: Yes
- Time schedule for examinations: on day 16 post coitum
- Parameters checked: nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, volume, secific gravity, sediment (fat, crystals, cells: renal tubular, transitional, sqamous, casts, erythrocytes, leucocytes, bacteria)

POST-MORTEM EXAMINATIONS: Yes (gross pathology)
- Sacrifice on gestation day 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead foetuses (hypoxemic foetuses which did not breath spontaneously after the uterus had beenopened): Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No

Statistics:

Dunett's Test was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of foetuses, weight of placentae, corpura lutea, implantations, pre- and postimplantation losses, resorptions, and live foetuses.
Fisher's Exact Test was used for statistical evaluation of conception rate, mortality of the dams, and all foetal findings.

Indices:

- conception rate (%) calculted by (number of pregnant animals / number of fertilised animals) x 100
- preimplantation loss (%) calculated by ((number of corpura lutea - number of implantations) / number of corpura lutea) x 100*
- postimplantation loss (%) calculated by ((number of implantations - number of live foetuses) / number of implantations) x 100

* calculation on the basis of each individual pregnant animal with scheduled sacrifice

Historical control data:

Historical control data were available.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
FERTILITY OF THE DAMS
The following number of females were not pregnant after the mating procedure:
- vehicle control group: 2/25
- 100 mg/kg bw/day group: 3/25
- 400 mg/kg bw/day group: 3/25
- 1000 mg/kg bw/day group: 1/25

CLINICAL SIGNS AND MORTALITY OF THE DAMS
- There were no mortalities in any of the groups throughout the study period.
- There were no abnormal clinical findings in any dam of any group throughout the study period.

FOOD CONSUMPTION OF THE DAMS
- The food consumption of all test groups (100, 400, and 1000 mg/kg bw/day) did not show any differences of biological relevance as compared to the control group. All values were within the range of biological variation.

BODY WEIGHT AND BODY WEIGHT GAIN OF THE DAMS
- the body weights and body weight gains of the dams of the test groups were similar to those of the control group. All differences observed between the groups were without any biological relevance.
- The result of the corrected body weight gain (terminal body weight on day 20 post coitum minus weight of the uterus before it was opened minus body weight on day 6 post coitum) of all substance treated groups did not show any differences of biological relevance if compared to the controls.

HAEMATOLOGY
No statistically significant changes were reported.

CLINICAL CHEMISTRY
No statistically significant changes were reported.

URINALYSIS
No statistically significant changes were reported.

NECROPSY OF THE DAMS
- The uterus weights of the animals were not influenced by test material administration. The differences between the groups were without biological relevance.

REPRODUCTION DATA OF THE DAMS
- The conception rate varied between 88% (100 and 400 mg/kg bw/day) and 96% (1000 mg/kg bw/day). There were no substance-related and/or statistically significant differences of biological relevance between the groups in conception rate, in the mean number of corpura lutea and implantation sites, in the values calculated for the pre- and postimplantation losses, or in the number of resorptions and viable foetuses. The differences evident were considered incidental and within the normal range of deviations for animals of this strain and age.

WEIGHT OF PLACENTAE
- The mean placental weights in the test groups were comparable to the actual control values.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
SEX DISTRIBUTION OF THE FOETUSES
- The sex distribution of the foetuses in the test groups was comparable with the control foetuses. The differences observed in comparison to the control were without any biological relevance.

WEIGHT OF FOETUSES
- The mean foetal weight were not influenced by the test substance administration. All values were within the range of biological variation.

EXTERNAL EXAMINATIONOF THE FOETUSES
- External malformations were recorded for one low dose foetus only (100 mg/kg bw/day). For this foetus, brachygnathia and aglossostomia were noted. These or very similar malformations were also present in the historical control data of the breeder at a low frequency. Therefore, and because these external malformations (in just one low dose foetus) show no relationship to dosing, they were finally assessed as being of spontaneous origin. No further variations were observed in the foetuses in any group at the external examination.
- So-called unclassified observations (placentae fused or necrobiotic) were recorded for 2 control, 4 low dose (100 mg/kg bw/day), and 2 mid dose foetuses (400 mg/kg bw/day), but not for any high dose foetus (1000 mg/kg bw/day).

SOFT TISSUE EXAMINATION OF FOETUSES
- The examination of the organs of the foetuses revealed one foetus with malformations in the low and mid dose each: hydrocephaly together with microphthalmia (left) and anophthalmia (right) occurred in one foetus of the low dose (100 mg/kg bw/day) and dextrocardia was recorded for one mid dose foetus (400 mg/kg bw/day). Due to the missing dose-response relationship and due to the fact that these malformations were also present in the historical control data, they were regarded as being of sponatneous nature and not related to test material administration.
- Variations (dilated renal pelvis and/or hydrourether) were detected in all groups without any statistically significant differences between the groups. All values were fully within the range of biological variation.
- No so-called unclassified observations (like blood coagulum araound the bladder) were recorded.

SKELETAL EXAMINATION OF THE FOETUSES
- Various skeletal malformations were seen in a number of foetuses of alll groups. These malformations were related to the skull (mandible fused or various skull abnormalities), the vertebral column (thoracic/lumbar vertebral body/bodies dumbell-shaped (asym.) or bipartite (asym.)), the sternum (sternebra(e) bipartite, ossification centers dislocated) and/or the ribs (fused ribs). Most of the described skeletal malformations occurred without any relation to dosing and without any statistically significant differences between the groups; however, the foetal and/or litter incidences for two malformations of the skeleton (thoracic/lumbar vertebral body/bodies dumbell-shaped (asym.); sternebra(e) bipartite, ossification centers dislocated) and the overall number of skeletal malformations were increased in the substance treated groups, but without a clear dose-response relationship. All values, however, are fully within or just above the historical control range. It is notable that the control values were unexpectedly low and that the relevant values for the substance-treated groups do show a clear dose-response relationship. Therefore, the statistically significanly, but not dose-relatedly, increased foetal and/or litter incidences concerning skeletal malformations were finally considered random.
- The variations elicitated were related to the ribs (shortened 13th, accessory 14th ribs or rudimentary cervical ribs), the vertebral column (accessory thoracic vertebra) and the sternum (sternebra(e) of irregular shape or bipartite) and were found in all groups without any dose-response relationship. The statitically significant increase of sternebra(e) of irregular shape (100 mg/kg bw/day) and the low occurrence of rudimentary cervical rib(s) (400 mg/kg bw/day) compared to controls were without any biological relevance; the diffrernces occurred without any relation to dosing and the values were fully within the range of the historical control.
- In all groups signs of retardations (incomplete or missing ossifications of vertebral bodies/arches, sternebra(e), skull and/or the hyoid bone) were found without any differences of biological relevance between the groups. The statistically significantly lower number of low dose foetuses (100 mg/kg bw/day) with sternebra(e) with only one ossification center and of high dose foetuses (1000 mg/kg bw/day) with not ossified sternebra(e) were of spontaneous nature; the relevant values were fully within the range ofthe historical control.


SUMMARY
In summary, there were no statistically significant differences between the control and the test groups concerning foetal external and soft tissue examinations. Only the evaluation of the foetal skeletons revealed statistically significant increase in the malformation rate in the low and mid dose groups (100 and 400 mg/kg bw/day), but not in the high dose group (1000 mg/kg bw/day). The statistically significant differences between the control and the low and mid dose groups were not test material related, but were of spontaneous nature due to unexpected low occurrence of dumbell-shaped (asym.) thoracic vertebral body/bodies and/or bipartite sternebra(e) with dislocated ossification centers in the control group. Moreover, the slightly, but statistically significantly increased number of low dose foetuses with skeletal variations was assessed as being of spontaneous nature due to missing dose-response relationship.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Tab. 1: Selected foetal skeletal malformations

			control	100 mg/kg bw/day	400 mg/kg bw/day	1000 mg/kg bw/day
Thoracic vertebral body/bodies dumbbell-shaped (asym.)	Foetal incidence	N	0	5*	10**	5*
		%	0.0	3.2	6.6	3.0
	Litter incidence	N	0	4*	7**	4
		%	0.0	18.0	32.0	17.0
Sternebra(e) bipartite, ossification center dislocated	Foetal incidence	N	0	1	4*	2
		%	0.0	0.6	2.6	1.2
	Litter incidence	N	0	1	3	2.
		%	0.0	4.5	14.0	8.3

statistical significance indicated as * p<0.05; ** p<0.01

Tab. 2: Incidence of selected skeletal malformations observed in the study compared to the historical control data

	Actual foetal incidence (%)		Historical foetal incidence (%)	Actual litter incidence (%)		Historical litter incidence (%)
Thoracic vertebral body/bodies dumbbell-shaped (asym.)	control 100 mg/kg bw/day 400 mg/kg bw/day 1000 mg/kg bw/day	0.0 3.2 6.6 3.0	0-8.8	control 100 mg/kg bw/day 400 mg/kg bw/day 1000 mg/kg bw/day	0.0 18.0 32.0 17.0	0-39.1
Sternebra(e) bipartite, ossification center dislocated	control 100 mg/kg bw/day 400 mg/kg bw/day 1000 mg/kg bw/day	0.0 0.6 2.6 1.2	0-2.2	control 100 mg/kg bw/day 400 mg/kg bw/day 1000 mg/kg bw/day	0.0 4.5 14.0 8.3	0-14.3

Applicant's summary and conclusion

Conclusions:

The test item had no effect on intrauterine development.