Registration Dossier

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: reproduction/developmental toxicity screening test

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003
Reference Type:
secondary source
Title:
No information
Author:
OECD SIDS
Year:
2005
Bibliographic source:
cited in OECD SIDS 1,3-Dimethylurea (CAS: 96-31-1)

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test substance : N,N'-Dimethylurea
- Test substance No.: 00/0445-1
- CAS No.: 96-31-1
- Batch No.: 67-0644
- Physical state/color: Solid/colorless
- Purity: 99.2g/100g
- Homogeneity: Homogeneous
- Stability: Proven by reanalysis after the in life phase of the study
- Storage conditions : Room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: (P) 76 - 83 days
- Weight at study initiation: (P) Males: 238 .7 (214.9 - 262.2) g; Females: 173 .0 (160.1 - 187.5) g
- Housing: individually
- Diet (e.g. ad libitum): Kliba laboratory diet rat/mouse/hamster (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: six days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
doubly distilled
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance solutions were prepared at a 4 day interval for the first time and thereafter daily. After the stability of the test substance in doubly distilled water was confirmed over a period of 10 days, the preparations were carried out at 10 day intervals.
Details on mating procedure:
- M/F ratio per cage: 1/1 (were put into the cages of the males)
- A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "day 0" and the following day "day 1" p .c. (post coitum).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration control analyses of the aqueous test substance solutions revealed that the values were in the expected range of the target concentrations (Mean values: 90.5% - 99 .8%). Thus, the correctness of the prepared concentrations was confirmed.
Samples were diluted with acetonitrile/doubly distilled water. Aliquots of the dilutions were used for HPLC-analysis.
Duration of treatment / exposure:
Exposure period: approx. 4 weeks
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: males: 4 weeks; females: until at least 4 days after giving birth
Frequency of treatment:
daily
Details on study schedule:
Acclimatization period: Jan. 9 - Jan. 14, 2001
Administration period: Jan. 15 - Feb . 12, 2001 (male animals); Jan. 15 - Mar . 1, 2001 (female animals)
Mating period for litter: Jan. 28 - Jan . 31, 2001
Gestation period: Jan. 29 - Feb . 22, 2001
Birth of litter: Feb . 19 - Feb . 23, 2001
Lactation period (including period after sacrifice of the litters): Feb . 19 - Mar . 2, 2001
Sacrifice of litter (on day 4 p. p.): Feb. 23 - Feb . 27, 2001
Sacrifice of parental animals: Feb. 13, 2001 (male animals); Mar. 2, 2001 (female animals)
Doses / concentrations
Remarks:
Doses / Concentrations:
0; 20; 60; 200 mg/kg bw/day in aqueous solution
Basis:
actual ingested
No. of animals per sex per dose:
10 animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
20 mg/kg body weight/day: as the expected no observed adverse effect level
60 mg/kg body weight/day: as the intermediate dose level and eventually as higher no observed adverse effect level
200 mg/kg body weight/day: as the dose level where toxic effects were expected
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: least once daily
- Cage side observations: dead or moribund animals, nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning); exceptions: During the mating period the parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 post coitum. Females with litter were weighed on the day of parturition (day 0 p.p.) and on days 4 and 7 post partum (if still alive and not sacrificed after day 4 p.p.). Females without litter were weighed weekly.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, once a week (in general for a period of 7 days) for male and female animals.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight
Litter observations:
PUP NUMBER AND STATUS AT DELIVERY: All pups derived from the F0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter. Pups, which died before the first determination of their status on the day of birth, were designated as stillborn pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, clinical symptoms (including gross-morphological findings), weight gain, necropsy observations.

GROSS EXAMINATION OF DEAD PUPS: yes, pups were examined externally, eviscerated and their organs were assessed macroscopically.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed about 4 weeks after the first test substance administration,
F1 pups were killed on day 4 post partum (p.p. ) and the F0 females on one of the following days.

GROSS NECROPSY
- Special attention was paid to the organs of reproductive system (testes, epididymides) and kidneys.

HISTOPATHOLOGY / ORGAN WEIGHTS: ovaries, testes, epididymides, kidneys, all gross lesions
Postmortem examinations (offspring):
SACRIFICE
- All surviving pups were sacrificed (by means of CO2) on day 4 p.p.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: These pups, all stillborn pups and those that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically . If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it deemed necessary, examined additionally using appropriate methods (e .g., skeletal staining according to modified DAWSON's method / Dawson, A. B ., 1926). The stained skeletons were evaluated under a stereomicroscope or a magnifying glass.

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No clinical signs which might be attributed to the test substance were detected in male or female F0 generation parental animals. The 3 doses (20 ; 60 ; 200 mg/kg body weight/day) administered by gavage did not lead to disturbances in general behavior in any of the F0 parental animals. One control male F0 parental animal showed chromodacryorrhea on its left eye nearly during the entire administration period (between days 0-9 and days 14-29) . This isolated finding in one control male is spontaneous in nature. There were no particular substance-related clinical findings in F0 females during the gestation period for F1 litter. One sperm positive female of 60 mg/kg body weight/day test group did not deliver any F1 pups . This is considered to be unrelated to treatment due to the isolated occurrence of this finding without a dose-response relationship. There were no substance-related or spontaneous clinical observations during lactation in any of the female F0 parental animals in any of the groups.
There were no substance-related mortalities in any of the male and female F0 parental animals in any of the groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): The food consumption of the high dose F0 male animals was statistically significantly
reduced during study weeks 0-1 and remained slightly reduced for the rest of the study period (weeks 1-4) without attaining statistical significance. Furthermore, the food consumption of the high dose females was also slightly reduced during premating and gestation, achieving statistical significance only for premating week 0-1 and gestation days 0-7 and 14-20 . During lactation (days 0-4 p .p.) food consumption of the high dose dams was similar to that of the controls. If calculated for the whole study period of the males (weeks 0-4), the food consumption of the high dose group was about 10% lower than the corresponding control value. Fu rthermore, the food consumption of the high dose females was about 8% lower during premating period (weeks 0-2) and about 7% lower during gestation days 0-20 p.c.. This was considered to be substance-related, because the slight reduction in food consumption is in-line with lowered mean body weight and impaired body weight gain of the high dose males and females. All other differences between the substance-treated and the concurrent control groups concerning food consumption are without any biological relevance and/or not doserelated. This includes the statistically significantly decreased food consumption of the 20 mg/kg males during premating weeks 1-2 and 3-4.
With the exception of the first week the mean body weights of the high dose male parental animals were statistically significantly decreased in comparison to the corresponding control values. At study week 4 the body weight of the high dose males was about 9% lower than in the controls. Furthermore, body weight gains of these animals were also impaired and attained statistical significance during study weeks 0-2, 3-4 and 0-4. If calculated for the entire treatment period, body weight gain of the high dose males was about 37% below the concurrent control value. Body weights/body weight gains of the F0 parental males of the 20 and 60 mg/kg groups revealed no substance-induced impairments . The isolated, but statistically significantly decreased mean body weight gain of the FO low dose males (20 mg/kg body weight/day) during weeks 1-2 was without any biological relevance.
The mean body weights of the high dose F0 females were were statistically significantly impaired during premating, gestation and lactation periods (about 6 - 9% below the corresponding control values at the end of the respective study phases). Body weight gains of these animals were also decreased and attained statistical significance during premating weeks 0-1 and 0-2, gestation days 0-7. If calculated for the different study phases, the F0 females gained about 53% less than the concurrent control group during premating, about 16% less during gestation and about 6% less during lactation. Body weights/body weight gains of F0 parental females in the 20 and 60 mg/kg body weight/day test groups were not influenced by the test substance administration neither during premating nor during gestation and lactation. All differences in body weights and body weight gains observed for these rats were without any biological relevance. This assessment includes the isolated, but statistically significantly decreased mean body weight gain of the F0 mid dose females during premating weeks 0-1. The described impairments in body weight data of the high dose males and females (200
mg/kg body weight/day), however, are considered to be substance-related and were accompanied by concurrent impairments in food consumption.

FEMALE REPRODUCTION AND DELIVERY DATA(PARENTAL ANIMALS): The female mating index calculated after the mating was 100% in all test groups (0; 20 ; 60 and 200 mg/kg body weight/day). The mean cohabitation time (duration until sperm was detected (i.e. day 0 p.c.)) amounted to 3.0 days/3.1 days/2 .5 days/2 .0 days (0 ; 20; 60 and 200 mg/kg body weight/day). These values reflect the normal range of biological variation inherent in the
strain used in this study. Consequently, the differences between the groups are assessed as spontaneous in nature and without any biological relevance. With the exception of one F0 parental female (test group 60 mg/kg body weight/day) all mated females (0 ; 20 ; 60; 200 mg/kg body weight/day) became pregnant. Therefore, the fertility indices varied between 90% and 100%. The fact that one mid dose female did not become pregnant is regarded to be spontaneous in nature and not associated with the treatment of the animals. There occurred no gross and/or histopathological findings in one mid dose female or its mating pa rtner, which could explain the observed infertility of this pair. The mean duration of gestation was very similar in all groups (between 21 .8 and 21 .9 days).
The gestation index was 100% for all groups, indicating that all pregnant females had live F1 pups in their litters. Implantation was not affected by treatment since the mean number of implantation sites was comparable between all test groups taking normal biological variation into account. Furthermore, there were no indications of substance-induced intrauterine embryo-/fetolethality since the postimplantation loss values were unaffected. The mean number of F1 pups delivered/dam was not affected by the test substance administered. The number of liveborn and stillborn pups was comparable between the groups, and the live bi rth index varied between 97% and 99%. Thus, the administration of N,N'-Dimethylurea did not adversely affect reproduction and delivery data of the F0 generation parental females.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental for reproductive performance and fertility
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

Details on results (F1)

PUP NUMBER AND STATUS AT DELIVERY (OFFSPRING): The mean number of delivered F1 pups/dam and the percentage of liveborn pups were not affected by the administration of the test substance. The differences observed are without any biological relevance and do not show a clear dose-response relationship.

VIABILITY (OFFSPRING): All pups except one of control dam died or were cannibalized on days 0-4 after delivery. Consequently, the viability index as indicator for pup mortality between days 0-4 p.p. was lower in the control group (84%) than in the substance-treated groups (i.e. 98%/99%/98% at 20, 60 or 200 mg/kg body weight/day). Thus, there are no substance-related differences concerning pup viability and mortality .

SEX RATIO (OFFSPRING): The sex distribution and sex ratios of live F1 pups on the day of birth and on day 4 p.p. did not show any substantial differences between controls and treated groups ; all observable differences are regarded to be spontaneous in nature.

CLINICAL SIGNS (OFFSPRING): None of the F1 pups showed any clinical signs.

BODY WEIGHT (OFFSPRING): Mean pup body weights/pup body weight gains did not show any statistically significant differences between the substance-treated groups and the concurrent control group. The observable fluctuations between the groups are without any biological relevance and are
related to the differences in litter size. Furthermore, the number of "runts" (i.e. stunted pups) did not show any differences between the groups for which a substance-induced origin could be assumed.

GROSS PATHOLOGY (OFFSPRING): The macroscopic examination of all pups at necropsy revealed only spontaneous findings at necropsy in a few of the large number of examined F1 pups . These findings were restricted to single pups of test groups 1 and 3 (20 and 200 mg/kg body weight/day). Macroglossia in combination with cleft palate occurred in three low dose pups from one litter and in one high dose pup. Two pups from the same high dose litter showed just cleft palate . As these findings are lacking a dose-response relationship, do also occur occasionally in control fetuses and/or pups of the strain of rats used for the present study and were restricted to only one litter each, they are considered to be spontaneous in nature and not induced by the test substance. A spontaneous background is also assumed for the two other gross necropsy observations, i.e. infarct of liver in one high dose pup and hemorrhagic testis in two low dose pups.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion