6-methyl-2-oxoperhydropyrimidin-4-ylurea

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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 similar to the OECD test guidelines 416 and 421, 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 Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

(the study focused on fertility/reproduction only and there was no 2nd generation)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

(the study focused on fertility/reproduction only and there was no 2nd generation)

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
- Substance type: white powder
- Physical state: solid
- Analytical purity: no data
- Batch No.: 1
- Expiration date of the lot/batch: not defined
- Storage condition of test material: at room temperature
- Date of receipt: 2002-03-15

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 Laboratories, L'Arbresle, France
- Age at study initiation: (P) 6 weeks
- Weight at study initiation: (P) Males: 171-210 g (mean: 192 g); females: 142-179 g (mean: 161 g)
- Housing: The animals were housed individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage. The cages were placed in numerical order on the racks. On a monthly basis, all the racks were moved clockwise around the room, rack by rack. In this way, for each group, identical exposure to environmental conditions was achieved.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: a 6-day acclimation period to the conditions of the study preceded the beginning of the treatment period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 °C
- Humidity (%): 50±20 %
- Air changes (per hr): about 12 cycles filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral 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. 101K0185, 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 60 and 120 mg/mL and then homogenised using an Ultraturrax laboratory mixer pending 5 minutes and a magnetic stirrer.
- The test substance dosage forms were made daily (preparation stable for 3 hours at room temperature).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and homogeneity were determined on samples of each control and test item dosage form prepared for use in week 1, 7 and 13. Three samples of each dosage form (from top, middle and bottom of the container) were taken on each occasion. They were stored at -20 °C pending dispatch (two consignments). The samples were sent on dry ice to the Sponsor. These analyses were carried out under the responsibility of the Sponsor.

Duration of treatment / exposure:

- males: throughout the pre-mating period (10 weeks), mating period (2 weeks), and until sacrifice;
- females: throughout the pre-mating period (10 weeks), mating period (2 weeks), and pregnancy until day 14 post-coitum inclusive.

Frequency of treatment:

daily 7 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
600 and 1200 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were specified by the Sponsor and based on the available toxicological data obtained in a combined repeat dose toxicity/developmental toxicity screening test with the test item: 600 mg/kg bw/day as the expected No Adverse Effect Level, 1200 mg/kg bw/day as themaximum feasible dose-level.
The oral route was selected since it is a possible route of exposure in human and it is requested by the regulatory authorities for this type of test item.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded once a week until sacrifice. The body weight of each female was recorded once a week during the pre-mating and mating periods, then on days 0, 7 and 15 post-coitum.

Sacrifice and pathology:

SACRIFICE
- males: all surviving animals after most hysterectomies of the females had been performed
- maternal animals: all surviving animals on Day 15 post coitum
- females which did not mate: at least one week after the end of the mating period

GROSS NECROPSY
- Gross necropsy consisted of a macroscopic post-mortem examination of the principal thoracic and abdominal organs (with particular attention paid to the reproductive organs) was performed on all animals, including those that died during the study or were killed prematurely. For all females, the number of corpora lutea and implantation sites was recorded whenever possible. Whenever necessary, photographs were taken to document findings and kept with the study archives.

HISTOPATHOLOGY / ORGAN WEIGHTS
No microscopic examination was deemed necessary since all the macroscopic lesions were considered to be unrelated to the treatment. The body weight of all animals killed at terminal sacrifice was recorded and the following organs were weighed (wet) as soon as possible after dissection: all males: testes, epididymides, prostate, seminal vesicles together with coagulating glands, pituitary gland and adrenals, testes and epididymides were weighed separately, all females: uterus, ovaries, pituitary gland and adrenals.

Other examinations:

- reproductive indices: mating data, male fertility data, female fertility data (female fertility and gestation indices, hysterectomy parameters)
- sperm parameters examined in [P] male parental generations: testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, and sperm morphology

Statistics:

Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances
being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

statisticallly significantly reduced in dams at 600 and 1200 mg/kg bw/day, no effect in males and in females before pregnancy. This effect is secondary to decreased food consumption.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

statisticallly significantly reduced in dams at 1200 mg/kg bw/day, no effect in males and females before pregnancy. This is considered to be based on abnormal taste and smell perception during pregnancy and as protective mechanism.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

No microscopic examination was deemed necessary since all the macroscopic lesions were considered to be unrelated to the treatment.

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY (PARENTAL ANIMALS)
- Males: No mortality was recorded in the control or the 1200 mg/kg/day groups. One male given 600 mg/kg/day was found dead on day 44. At macroscopic post-mortem examination, esophagus perforation was noted together with serous contents in the thoracic cavity and whitish deposit on the pleura. This death was considered of accidental nature and without any relationship to treatment with the test item.
- Females: There was no death, in any control or treated group over the treatment period.

CLINICAL SIGNS (PARENTAL ANIMALS)
- Males: The few clinical signs recorded in three males given 600 mg/kg/day (piloerection or chromodacryorrhea and chromorhinorrhea) and one male given 1200 mg/kg/day (ptyalism) were not considered to be of toxicological importance since the incidence of these findings was low and not dose-related.
- Females: The few clinical signs (ptyalism) recorded in one female given 600 mg/kg/day and three females given 1200 mg/kg/day did not represent an adverse effect. Other observations (area of hair loss on the forelimb or hindlimb) recorded in some males and females are among findings commonly observed in rats of this strain and age.

FOOD CONSUMPTION AND BODY WEIGHT (PARENTAL ANIMALS)
- Males: The food consumption of the treated males was similar to that of the control group.
- Females: During pre-mating (days 1 to 71), the food consumption was similar in the control and the treated groups. During pregnancy (days 1 to 15), minimal (-7%) and slight (-14%, p<0.01) decreases in food consumption were recorded at the 600 and 1200 mg/kg/day dose-levels, respectively.

- Males: There was a minimal non significant decrease in body weight gain in males given 600 (-6%) and 1200 mg/kg/day (-5%) over the treatment period (days 1 to 92). Because these minor changes in body weight gain were not statistically different, not dose-related and did not correlate with any change in food consumption, they were considered to be marginal and of no toxicological significance.
- Females: During the pre-mating period (days 1 to 71), the body weight gain at 600 mg/kg/day was similar to that of the controls, while it was slightly lower (-11 %, p<0.05) in females given 1200 mg/kg/day. During pregnancy (days 1 to 15), a similar moderate decrease in body weight gain was recorded in females given 600 (-24%, p<0.001) and 1200 mg/kg/day (-24%, p<0.001). These changes on body weight gain and food consumption were considered to be not adverse, since the effect was observed in pregnant females solely and did not affect the reproductive outcome.

Expert judgement:
These changes on body weight gain and food consumption were considered to be not adverse, since the effect was observed in females solely, and not before pregnancy. It is generally known, that abnormal taste and smell perception is observed during pregnancy. It can therefore be expected that reduced food consumption is a consequence of abnormal taste and smell during gestation and may be a foeto-protective mechanism to avoid poisoning. This protective mechanism is expected to be enhanced by treatment with the test item, as a dose relationship was observed. In conclusion, the decreased body weight gain during pregnancy is considered to be more a gestation-related effect rather than a test-material related effect. Moreover, the decreased body weights did not affect the offspring, thus supporting that this observation can be concluded as non-adverse.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The minimal fluctuations recorded in the seminology parameters in the treated groups were not dose-related, not notably different from the controls, and in the range of CIT historical control data. Consequently, they were not considered of toxicological significance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
When compared with their respective controls, higher absolute and relative adrenal weights were observed for treated males and lower absolute and relative ovary and uterus weights were noted for treated females. Considering that the differences in the adrenal weights were not dose-related and observed in the males only, they were considered to be without relationship to the treatment.
The differences in the weights of the reproductive organs, particulary for uterus and ovaries were considered not to be treatment-related. Indeed, most of individual values of ovaries weight at 600 and 1200 mg/kg/day were within the range of control group values. Concerning the slight decrease observed in uterus weight, this latter change was only due to the contribution of a few individual values of two females. In addition, the differences (% from control group) in the ratio of uterus weight/number of foetuses and of ovaries weight/number of corpora lutea between the treated and control animals was minor and not dose-related. In conclusion, these minor differences from the control group in ovaries and uterus weights were considered not to be related to the treatment with the test item.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The few macroscopic findings recorded were those, which are commonly observed spontaneously in the untreated laboratory rat of this strain and age and thus considered to be of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
MORTALITY (PARENTAL ANIMALS)
- Males: No mortality was recorded in the control or the 1200 mg/kg/day groups. One male given 600 mg/kg/day was found dead on day 44. At macroscopic post-mortem examination, esophagus perforation was noted together with serous contents in the thoracic cavity and whitish deposit on the pleura. This death was considered of accidental nature and without any relationship to treatment with the test item.
- Females: There was no death, in any control or treated group over the treatment period.

CLINICAL SIGNS (PARENTAL ANIMALS)
- Males: The few clinical signs recorded in three males given 600 mg/kg/day (piloerection or chromodacryorrhea and chromorhinorrhea) and one male given 1200 mg/kg/day (ptyalism) were not considered to be of toxicological importance since the incidence of these findings was low and not dose-related.
- Females: The few clinical signs (ptyalism) recorded in one female given 600 mg/kg/day and three females given 1200 mg/kg/day did not represent an adverse effect. Other observations (area of hair loss on the forelimb or hindlimb) recorded in some males and females are among findings commonly observed in rats of this strain and age.

FOOD CONSUMPTION AND BODY WEIGHT (PARENTAL ANIMALS)
- Males: The food consumption of the treated males was similar to that of the control group.
- Females: During pre-mating (days 1 to 71), the food consumption was similar in the control and the treated groups. During pregnancy (days 1 to 15), minimal (-7%) and slight (-14%, p<0.01) decreases in food consumption were recorded at the 600 and 1200 mg/kg/day dose-levels, respectively.

- Males: There was a minimal non significant decrease in body weight gain in males given 600 (-6%) and 1200 mg/kg/day (-5%) over the treatment period (days 1 to 92). Because these minor changes in body weight gain were not statistically different, not dose-related and did not correlate with any change in food consumption, they were considered to be marginal and of no toxicological significance.
- Females: During the pre-mating period (days 1 to 71), the body weight gain at 600 mg/kg/day was similar to that of the controls, while it was slightly lower (-11 %, p<0.05) in females given 1200 mg/kg/day. During pregnancy (days 1 to 15), a similar moderate decrease in body weight gain was recorded in females given 600 (-24%, p<0.001) and 1200 mg/kg/day (-24%, p<0.001). These changes on body weight gain and food consumption were considered to be related to treatment with the test item.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The minimal fluctuations recorded in the seminology parameters in the treated groups were not dose-related, not notably different from the controls, and in the range of CIT historical control data. Consequently, they were not considered of toxicological significance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The female mating index (mated/paired) and pre-coital interval were similar in all groups. Most paired animals mated within 1 to 4 days of cohabitation except for a few pairs in each group, for which the duration was slightly longer. This finding, commonly recorded at this low incidence was not attributed to treatment with the test item. The slight fluctuations in male fertility parameters, which were not dose-related, are commonly recorded in rats of this strain and age, consequently, they were not considered to be related to the treatment with the test item.
The treatment with the test item did not disturb fertility and gestation indices of the females at any dose-level. The number of corpora lutea was minimally lower in the treated groups when compared to the controls. Since the difference was small, not statistically significant, and within the range of CIT historical control data, these findings were considered to have occurred by chance. Hence, at 600 and 1200 mg/kg/day the number of implantation sites and concepti was also lower than the control, gaining statistical significance only at-the top dose-level. Since these values were also within CIT historical control data, they were considered to be a consequence of the fortuitous lower number of corpora lutea rather than a test item related effect.
The slight fluctuations in the pre- and post-implantation losses were not considered to be related to the treatment with the test item since they were not dose-related and/or not significantly different from either the concurrent control or the CIT historical control data.
ORGAN WEIGHTS (PARENTAL ANIMALS)
When compared with their respective controls, higher absolute and relative adrenal weights were observed for treated males and lower absolute and relative ovary and uterus weights were noted for treated females. Considering that the differences in the adrenal weights were not dose-related and observed in the males only, they were considered to be without relationship to the treatment.
The differences in the weights of the reproductive organs, particulary for uterus and ovaries were considered not to be treatment-related. Indeed, most of individual values of ovaries weight at 600 and 1200 mg/kg/day were within the range of control group values. Concerning the slight decrease observed in uterus weight, this latter change was only due to the contribution of a few individual values of two females. In addition, the differences (% from control group) in the ratio of uterus weight/number of foetuses and of ovaries weight/number of corpora lutea between the treated and control animals was minor and not dose-related. In conclusion, these minor differences from the control group in ovaries and uterus weights were considered not to be related to the treatment with the test item.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The few macroscopic findings recorded were those, which are commonly observed spontaneously in the untreated laboratory rat of this strain and age and thus considered to be of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No microscopic examination was deemed necessary since all the macroscopic lesions were considered to be unrelated to the treatment.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

LOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduced body weight gain

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Additional data:

ORGAN WEIGHTS:

- females: significantly lower absolute and relative ovary and uterus weights (ovaries: low, high-dose/absolute: -12, -13%; low, high-dose/relative: -7, -4% as compared to controls; uterus: low, high-dose/absolute: -19, -21%; low, high-dose/relative: -14, -12% as compared to controls). These differences were considered not to be treatment related, as most of the values were within the range of the control group, and changes were only due to the contribution of a few individual values of two animals.

- males: significantly, but not dose-related higher absolute and relative adrenal weights (low, high-dose/absolute: +24, +18%; low, high-dose/relative: +29, +23% as compared to controls). Because of the lack of a dose-response and because only seen in males, these effects were considered as of no biological significance.

 

MATING DATA:

The female mating index (mated/paired) and pre-coital interval were similar in all groups (control, low, high-dose: female mating index 96 - 96 - 96%, mean pre-coital interval 2.7 - 2.8 - 3.2 days).

 

MALE FERTILITY DATA:

The male mating index and the male fertility index were similar in all groups (male mating index: control, low, high-dose: 100 - 96 - 96 %, male fertility index: 100 - 91.3 - 91.3 %).

 

FEMALE FERTILITY DATA:

The following was obtained for the control, low, and high-dose groups, respectively:

- mated females: 24, 24; 24

- pregnant females: 24, 21, 22

- female fertility index (%): 100, 87.5, 91.7

- females with live concepti: 24, 21, 22

- gestation index (%): 100, 100, 100

 

NUMBER OF CORPORA LUTEA, NUMBER OF IMPLANTATIONS (control, low, high-dose groups):

- corpora lutea: 17.6, 16.4, 16.0

- implantation sites: 16.6, 14.9, 14.5* (p<0.05)

- pre-implantation loss (%): 5.7, 9.6, 9.1

- concepti: 15.2, 14.3, 13.0* (p<0.05)

- post-implantation loss (%): 8.3, 4.2, 11.0

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

The female mating index (mated/paired) and pre-coital interval were similar in all groups. Most paired animals mated within 1 to 4 days of cohabitation except for a few pairs in each group, for which the duration was slightly longer. This finding, commonly recorded at this low incidence was not attributed to treatment with the test item. The slight fluctuations in male fertility parameters, which were not dose-related, are commonly recorded in rats of this strain and age, consequently, they were not considered to be related to the treatment with the test item.

The treatment with the test item did not disturb fertility and gestation indices of the females at any dose-level. The number of corpora lutea was minimally lower in the treated groups when compared to the controls. Since the difference was small, not statistically significant, and within the range of CIT historical control data, these findings were considered to have occurred by chance. Hence, at 600 and 1200 mg/kg/day the number of implantation sites and concepti was also lower than the control, gaining statistical significance only at-the top dose-level. Since these values were also within CIT historical control data, they were considered to be a consequence of the fortuitous lower number of corpora lutea rather than a test item related effect.

The slight fluctuations in the pre- and post-implantation losses were not considered to be related to the treatment with the test item since they were not dose-related and/or not significantly different from either the concurrent control or the CIT historical control data.