6-methyl-2-oxoperhydropyrimidin-4-ylurea

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance 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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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

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

Constituent 2

Reference substance name:

isobutylidene diurea

IUPAC Name:

isobutylidene diurea

Details on test material:

- Name of test material (as cited in study report): isobutylidene-di-urea
- Substance type: powder
- Physical state: solid
- Analytical purity: 90.1%
- Batch No.: 1/1/98
- Storage condition of test material: at room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: males: 8 weeks; females: 10 weeks
- Weight at study initiation: males: 276 g mean bw; females: 228 g mean bw
- Housing: housed individually in suspended wire-mesh cages, females shifted 4 days before lactation to polycarbonate cages in a barrier rodent unit.
- Diet: A04 C pelleted diet ad libitum, distributed weekly
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21± 2
- Humidity (%): 50±20
- Air changes (per hr): about 12 cycles of filtered, non recycled air.
- 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 vehicle was 0.5% aqueous carboxymethylcellulose solution prepared using:
- purified water, obtained by reverse osmosis using a Milli-Ro 8 plus apparatus (Millipore SA, Saint-Quentin en Yvelines, France).
- carboxymethylcellulose, batch No. 69H0028, supplied by Sigma (Saint-Quentin-Fallavier, France).

The test substance was administered as a suspension in the vehicle.
The test substance was ground using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 10, 30 and 100 mg/mL
and then homogenized using a magnetic stirrer.
The test substance dosage forms were made daily (preparation stable for 3 hours) .

Details on mating procedure:

- M/F ratio per cage: 1 male/1 female of the same dose group
- Length of cohabitation: during the night
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- Each female was placed with the same male until mating occured or 14 days had elapsed.

Analytical verification of doses or concentrations:

yes

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 .
They were stored at -20°C pending dispatch, to the Sponsor, for analysis of concentration and homogeneity

Duration of treatment / exposure:

Exposure period: males: pre-mating, mating (14days) and post-mating, for a total of 34 days, females: pre-mating, mating, pregnancy and lactation until day 4 post partum
Premating exposure period (males): 15 days
Premating exposure period (females): 15 days
Duration of test: males: until sacrifice in the post-mating period, females: until day 4 post partum

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

no futher data

Doses / concentrations

Remarks:

Doses / Concentrations:
100; 300; 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were specified by the Sponsor, following the results of a prev iously conducted prenatal developmental
toxicity study in Wistar rats (BASF Project No. 30R0727/90116). The oral route was selected since it is the route of exposure, which is requested by
regulatory authorities for this type of product.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily at least once

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first day of treatment and then once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: male animals: first day of treatment, then once a week until sacrifice; female animals: first day of treatment, then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 1 and 4 post-partum

FOOD CONSUMPTION AND COMPOUND INTAKE :
The 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 .

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: testis weight, epididymis weight, morphological examination of the testes: tailed and round spermatids, spermatocytes, spermatogonia, different stages of spermatogonic cycle

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
-The following parameters were examined in [F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external abnormalities; possible cause of death was not determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the completion of the treatment period: after the end of the mating period: on study day 35 (total treatment period was 34 days)
- Maternal animals: All surviving animals after the completion of the treatment period: the females and their pups were killed on day 5 post partum. Female animals showing no evidence of mating were killed 24-26 days after the last day of mating.

GROSS NECROPSY
- 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. 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
- body weight and organ weights were examined (adrenals, brain, heart, kidneys, liver, spleen, thymus; additionally in males: testes and epididymides; females:ovaries)
- Histopathology: macroscopic lesions, adrenals, brain, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, sternum, stomach, testes, thymus, thyroids and parathyroids, trachea, urinary bladder, uterus

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations: externally for gross abnormalities, pup body weight, sex ratio, clinical signs

Statistics:

Dunnett, Fisher`s exact, Dunn, Mann-Whitney, Wilcoxon tests.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower bw gain at 1000 mg/kg bw in females during gestation and lactation; no effect in males and in females before pregnancy. The lower bw gain did not affect reproductive outcome and offspring and was therefore considered as not adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower bw gain at 1000 mg/kg bw in females during gestation and lactation; no effect in males and in females before pregnancy. The lower bw gain did not affect reproductive outcome and offspring and was therefore considered as not adverse.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Accumulation of the sex-linked alpha-2-µ-globulin was noted in 300 and 1000 mg/kg bw/day treated males. This effect is not of toxicological relevance for humans.

Other effects:

no effects observed

Description (incidence and severity):

Test substance intake: At 1000 mg/kg bw/day slightly lower food consumption in females during gestation, no effect in males and females before pregnancy. The lower food consupmtion did not affect reproductive outcome and offspring and was therefore considered as not adverse.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The reduced mating index was not correlated to pathologic changes in the reproductive organs and thus considered accidental.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No deaths occurred in any group, which were attributed to treatment with the test substance. In the male and female treated groups, there were no clinical signs, which were considered to represent adverse effect of the test substance in any animal.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Males: The food consumption of all the treated males was similar to that of the control males, during the pre-mating period. The body weight change of the treated males was similar to that of the control animals.
- 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). This difference correlating with an effect on body weight gain was considered to be related to treatment with the test substance. The body weight change of the females given 100 or 300 mg/kg bw/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. These differences, correlating with a reduction of food consumption, (during the pregnancy period) was considered to be not adverse, since the effect was observed in pregnant females solely and did not affect the reproductive outcome and the offspring.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The pre-coital interval was similar in the control and the treated groups. All paired animals (except one control pair) mated within 1 to 4 days of cohabitation, i .e. within the duration of a single estrous cycle. No particular microscopic finding was noted in the treated ovaries as their morphological characteristics were normal (particularly the number of ovarian follicles and corpora lutea). Hyalinosis of the blood vessels, haemosiderosis and sometimes capillary haemorrhage, were seen with similar incidence and/or severity in the uterus of the treated and control females.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treament-related abnormalities were found in the treated animals. The tailed and round spermatids were unaffected and the different stages of spermatogenic cycle were undisturbed by treatment with the test substance. In almost all treated as well as control males, a few degenerated necrotic cells sloughed in the lumen (minimal or slight), multinucleated giant cells (minimal), vacuoles of the seminiferous tubules (minimal or slight) were noted with similar incidence and/or severity in all control and treated groups. These findings with such severity are commonly recorded as spontaneous changes in the rat and were considered to be of no toxicological importance. The seminiferous tubules were lined with Sertoli cells only (minimal or slight) in 1/10 males given 300 mg/kg bw/day and in 2/10 males given 1000 mg/kg bw/day. For a third male from the same group, tubules lined with Sertoli cells only were considered to be tubuli recti as they were situated beneath the capsule. For 1 /10 males given 300 mg/kg bw/day and anothergiven 1000 mg/kg bw/day, minimal reduction in the number of spermatids was observed in very few seminiferous tubules. Minimal vacuolization of Sertolicells was observed in 1/10 males given 1000 mg /kg bw/day. Although not found in the control males, these microscopic abnormalities recorded with minimal severity in few or very few seminiferous tubules of a few males were considered to be without relationship to the treatment and most probably fortuitous.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The mating index was 100% for the pairs of the control, 100 and 300 mg/kg bw/day groups. The mating index was lower in the 1000 mg /kg bw/day group: 7/10 pairs (70%) mated within the two weeks of cohabitation. As no treatment-related morphological changes were noted in the genital organs of the high-dose male and female animals, this finding is considered to be accidental. The fertility index (pregnant/mated) was similar in the control and the treated groups, ranging from 90 to 100% without indication of dose- or treatment-relationship.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No compound-related differences in organ weights were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The few macroscopic observations recorded were those commonly encountered in the laboratory rat of this strain and age and thus considered to be of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There appears to be a dose-related higher severity of acidophilic globules in the cortical tubular epithelium of the kidneys of the treated male rats, given 300 and 1000 mg/kg bw/day. As 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 2 micro-globulin in the cortical tubular cells of the male rats. This is a sex- and species-specific effect, as no such accumulation occurs in female rats or in humans. Thus, this finding has no relevance for humans in terms of risk assessment.

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: histopathology: higher severity of acidophilic globules in the kindneys due to accumulation of alpha-2-u-globulin. As a sex-and species-specific effect of male rats, this finding has no relevance for humans

2

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduced food consumption and body weight gain in dams during gestation and lactation

3

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: taking into account that alpha-2-u-globulin accumulation in male rats is not relevant for humans and body weight gain reduction in females did not affect the reproductive outcome

4

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The number of pups which died during the four days of observation after birth was low (0 to 5 %) and similar in all the groups.

CLINICAL SIGNS (OFFSPRING)
There were no notable clinica l signs in the pups of the control or treated groups after birth.

BODY WEIGHT (OFFSPRING)
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) .

GROSS PATHOLOGY (OFFSPRING)
There were no gross external abnormalities in the control and the treated groups.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The test item had no effect on reproductive performance.