Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Urea, as indicated in the substance composition.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 100116B3 obtained from Borealis Agrolinz Melamine GmbH
- Expiration date of the lot/batch: January 10, 2017
- Purity test date: 11 July 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, dry and protected from light.
- Stability under test conditions: expiry date covered till January 10, 2017
- Solubility and stability of the test substance in the solvent/vehicle: stability is tested in the study
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: dilution of solid in tap water
- Final preparation of a solid: dilution in tap water

FORM AS APPLIED IN THE TEST (if different from that of starting material): dilution in tap water.

Test animals

Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at day 0 of pregnancy: 59 days
- Weight at day 0 of pregnancy: 199.8 - 251.0 g
- Fasting period before study: Not applicable.
- Housing: Except during the mating period, the dams were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were cleaned and changed once a week. The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages. Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
- Diet (e.g. ad libitum): Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany served as food. This food was offered daily ad libitum.
- Water (e.g. ad libitum): Drinking water (in drinking bottles) was offered ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%):55% ± 15% (maximum range)
- Air changes (per hr): between fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From (Start of mating) August 15, 2016 To: September 08, 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: tap water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared every day.
The test item was dissolved in the vehicle to the appropriate concentration and was administered orally at a constant volume (5 mL/kg bw/day ) once daily from the 6th to the 20th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.
The male rats for mating remained untreated.

VEHICLE
- Justification for use and choice of vehicle (if other than water): tap water

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approximately 4 mL were taken (divided into 2 aliquots of 2 mL) at the following times and stored at -20°C or colder until analysis at LPT.
1) At start of dosing:
Analysis of stability and concentration:
Immediately after preparation of the formulations as well as after 8 and 24 hours storage of the test item preparations at room temperature.
3 samples/dose level group; groups 2 - 4
Number of samples: 3 x 3 = 9
Sampling date: August 23, 2016

2) At the end of the dosing period (at a time when the majority of animals was dosed):
Analysis of concentration:
During treatment with the test item always before administration to the last animal of the dose level group.
1 sample/dose level group; groups 2 – 4
Number of samples: 1 x 3 = 3
Sampling date: September 07, 2016

Sum of all samples: 12

The samples were labelled with the study number, species, type of sample, aliquot number, concentration, sampling time and date.
The analytical method (HPLC-UV) was validated by LPT. The following parameters were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specifity
- Stability.
The investigation of the validation parameters (see above) indicated that the method employed is suitable for the determination of Urea in test item formulations.
The results of the test item formulation analysis are as follows:
-Concentration and stability before administration to the last animal on test day 6: 101.0% - 105.8% of nominal concentration
-Concentration before administration to the last animal on test day 21: 102.4% - 103.6% of nominal concentration.

The measured actual concentrations of the test item in the test item vehicle mixtures were between 102.4% and 105.8% of the nominal concentrations, indicating correctly prepared formulations.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm.
- Further matings after two unsuccessful attempts: yes. If findings were negative (no sperm in vaginal smear), mating was repeated with the same partner. This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:
Day 6 to 20 of gestation
Frequency of treatment:
once daily
Duration of test:
22 days:
- 5 adaptation days
- 1 mating day
- 15 administration days from gestation days 6 to 20
- Laparotomy on gestation day 21
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
based on test item
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
based on test item
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
based on test item
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
based on test item
No. of animals per sex per dose:
25 pregnant females per group
20 litters per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale in rats from literature:
Urea was shown to be of low toxicity following chronic administration to the rat. In a 12-month carcinogenicity screening assay, F344 rats (50/sex/group) were exposed to urea in the diet at concentrations of 4500, 9000 or 45000 ppm for 12 months. Five animals/sex/group were sacrificed at the end of the 365-day exposure for histopathology; interim deaths were similarly investigated. All remaining animals were sacrificed after the 4-month recovery period and investigated histopathologically. There were no signs of toxicity. Survival and body weights were unaffected by treatment. Gross and microscopic pathology did not reveal any treatment-related effects. No effects were observed at the highest dose level (4.5% in the diet). It is therefore concluded, based on the results of this study, that urea is of very low chronic toxicity (Fleischman et al., 1980).
Limited information on urea was available from an older non-guideline study in rats. Female rats (6/group) were gavaged with urea at doses equivalent to 500 mg/kg b.w./day during gestation for 14 days. Pups were necropsied at 48 hours after delivery and assessed for teratogenicity and renal effects. No hypertrophy or other kidney changes were detected in the pups, nor were there any teratogenic effects noted. Pup weight at birth was slightly lower in the treated group (5.15 g) compared to controls (5.93 g), but pups from treated dams gained more weight over the study period (Seipelt et al., 1969).
References:
-Fleischman, R.W., Baker, J.R., Hagopian, M., Wade, G.G., Hayden, D.W., Smith, E.R., Weisburger, J.H. and Weisburger, E.K.: Carcinogenesis bioassay of acetamide, hexanamide, adipamide, urea and P-tolylurea in mice and rats. Journal of Environmental Pathology and Toxicology 3(5-6): 149-70, 1980.
-Seipelt, H., Zoellner, K., Hilgenfeld, E. and Grossmann, H.: Studies on kidneys of newborn rats after chronic urea administration to the mother. Zeitschrift fur Urologie und Nephrologie 62(8): 623-7, 1969.
Based on the outcome of these two studies, it was decided to test up to the highest dose of 1000 mg/kg bw (limit dose) in a dose range finding study for a prenatal developmental toxicity. The results of this dose-range-finding study (LPT study no. 33779) showed a slightly reduced body weight for the dams of the high dose group (1000 mg/kg bw), but no changes were noted in behaviour or external appearance and no noteworthy differences in food consumption were noted. Reproduction data per group and per dam and fetal/placental weights did not show any differences between groups.
Together with the fact that no external malformations or variations were noted for the fetuses, dose levels of 100, 300 or 1000 mg Urea/kg bw/day were selected in agreement with the Sponsor for the present main study (LPT study no. 33780).

- Rationale for animal assignment (if not random):
Four (4) groups of pregnant rats were established, each obtained from matings which were carried out on a daily basis. Vaginal lavages were taken each morning. Day 0 of pregnancy was the day on which sperm was found in the vaginal lavage. When positive, the animals were assigned to the test groups by mating day using a Provantis®-generated randomization based on the body weight of the animals.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (for any signs of behavioural changes, reaction to treatment, or illness.)
Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared.
In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion occurred in the study.

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: /

BODY WEIGHT: Yes
- Time schedule for examinations:
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weightings - always at the same time of the day.
The body weight gain was calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight change from day 6 are given.
Carcass weight = Terminal body weight - uterine weight
Net weight change from day 6 = Carcass weight - body weight on day 6
These measurements were also used for calculating the daily amount of test item to be administered.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.
The relative food consumption (g/kg bw/day) was calculated using the following formula:
Daily food consumption [g/kg bw/day]= Total food intake in g /Body weight in kg
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No; not applicable (gavage)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. Dehydration of the animals was avoided.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 (CO2 narcosis)
- Organs examined: ovaries, uterus, internal organs and placenta.
The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations.
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
- Other: live/dead fetuses; sex ratio of fetuses; macroscopic inspection of the placentae for example for focal indurations or abnormal appearance; placentae weights and fetuses weights
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external or internal macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
Note:
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group was done using StatXact 4.0.1 software.
Indices:
Group indices:
Pre-implantation loss [%] = [Corpora lutea (per group) - Implantations (per group)/ Corpora lutea (per group)]x 100
Post-implantation loss [%] = [Implantations (per group) - living fetuses (per group)/ Implantations (per group)] x 100

Mean indices per litter:
Pre-implantation loss [%] = sum of pre-implantation losses per litter in a group [%]/ number of litters in a group
Post-implantation loss [%] = sum of post-implantation losses per litter in a group [%]/ number of litters in a group
Historical control data:
Summarized results of the 59 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH) performed at CRO in the years 2000 to July 2015

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No changes in behaviour, the external appearance and the faeces were noted in the control group and in the treatment groups.
Mortality:
no mortality observed
Description (incidence):
No premature death was noted in the control group and in the test item-treated groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related changes in body weight in comparison to the control group were noted for the dams of the treatment groups.
The values of body weight gain showed no differences between the control group and the test item-treated groups for the period after the start of treatment (gestation days 6 to 21).
A marginal but statistically significant difference (at p ≤ 0.05) compared to the control was noted for the body weight gain of the high dose group between gestation days 0 to 3. This finding is considered to be incidental due to its occurrence before start of the treatment period on gestation day 6.
No test item-related differences for the absolute body weight gain and the net body weight gain (without gravid uterus) between the control group and the dams of the treatment groups were noted between gestation day 6 and gestation day 21.
The marginally decreased net body weight gain (without gravid uterus) in comparison to the control (statistically not significant) noted for the dams of the intermediate dose group (minus 15.3%) and the high dose group (minus 18.5%) is within the range of normal variability and not considered to be test item-related or adverse.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related differences in food consumption were noted between the dams of the control group and the dams treated with 100, 300 or 1000 mg Urea/kg bw/day.
An incidental increase in relative food consumption was noted for the low dose group on gestation days 3 to 4, before start of the treatment period on gestation day 6, being statistically significant (at p ≤ 0.01) compared to the control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related differences were noted between the gravid uterus weight and carcass weight of the control dams and the dams of the treatment groups.
The placental weights showed no test item-related differences between the control group and the treatment groups.
At 300 mg Urea/kg bw/day, a slight but statistically significant decrease (at p ≤ 0.05) was noted for the mean placental weights (mean: 0.49 g) compared to the incidentally high control values (mean: 0.55 g). This finding is considered as not test item-related as the values were within the range of LPT background data (0.48 - 0.53 g). Above all, no corresponding findings were noted for the high dose group (1000 mg Urea/kg bw/day).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic inspection of the internal organs and tissues of the dams during laparotomy revealed no test item-related changes in the dams of the control and the dams of the treatment groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations. Macroscopic inspection of the internal organs and tissues of the dams during laparotomy revealed no test item-related changes in the dams of the control and the dams of the treatment groups.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortion occurred in the study.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation) were noted between the dams of the control group and the dams of the treatment groups.
No dams with total implantation loss were seen in the control and treatment groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation) were noted between the dams of the control group and the dams of the treatment groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation) were noted between the dams of the control group and the dams of the treatment groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
No abortion occurred in the study and there were no dams without viable fetuses in the control and treatment groups.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): OECD 414. There were no dams with early delivery observed in the control and treatment groups.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One animal in the 100 mg/kg group was not pregant; no other non-pregancies were observed.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The fetal weights showed no test item-related differences between the control group and the treatment groups.
Fetuses were considered as runts if their weight was less than 70% of the mean litter weight. No runts were noted at 100, 300 or 1000 mg Urea/kg bw/day.
One runt was noted in the control group (no. 13-13). This incidence is within the normal range of variation.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The fetal weights showed no test item-related differences between the control group and the treatment groups.
Fetuses were considered as runts if their weight was less than 70% of the mean litter weight. No runts were noted at 100, 300 or 1000 mg Urea/kg bw/day.
One runt was noted in the control group (no. 13-13). This incidence is within the normal range of variation.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the test item treated groups (100, 300 or 1000 mg Urea/kg bw/day).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related differences between the ratio of male and female fetuses (range: 0.90 - 1.12) were noted between the control group and the treatment groups.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
Description (incidence and severity):
Not applicable to OECD 414 study design.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopically visible external alterations (malformations or variations) were noted for the fetuses of the treatment groups (100, 300 or 1000 mg Urea/kg bw/day) during the macroscopic inspection at laparotomy.

Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (100, 300 or 1000 mg Urea/kg bw/day) during the skeletal examination according to DAWSON.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No malformations were noted for the fetuses of the control group and the fetuses of the treatment groups (100, 300 or 1000 mg Urea/kg bw/day) during the soft tissue examination according to WILSON.
During the examination of the organs and tissues according to WILSON variations were noted for the cerebrum ((subdural) haemorrhage), the cerebral ventricle (dilatation of the 4th ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis, reduced in size or malpositioned) and the liver (haemorrhagic focus/foci).
No test item-related differences in the incidences of the observed variations were noted between the control group and the treatment groups.
The slightly but significantly increased incidence of total soft tissue variations (at p ≤ 0.05 compared to the control) noted for the intermediate dose group (300 mg Urea/kg bw/day) is considered to be not test item-related as no dose-response relationship was noted and the incidence was within the range of LPT background data.
Description (incidence and severity):
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the treatment groups (100, 300 or 1000 mg Urea/kg bw/day).
Skeletal variations were noted for the ribs (wavy) and the sternebrae (bipartite, misaligned to a slight degree or misshapen).
No test item-related increase in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (100, 300 or 1000 mg Urea/kg bw/day).
Retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal and/or interparietal areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped), the sacral vertebral bodies (unossified), the caudal vertebral bodies (no or only one body ossified), the os pubis and the os ischii (in-completely ossified or unossified) and the metacarpalia and metatarsalia (absence of ossification in metacarpalia 2 to 5 and metatarsalia 2 to 5).
No test item-related increase in the incidence of skeletal retardations at 100, 300 or 1000 mg Urea/kg bw/day was noted during skeletal examination according to DAWSON.
A thoracic cavity filled with blood was noted for one fetus each of the control group and the low dose level group and two fetuses of the high dose group. These observations are a preparation induced artefact and not considered as test item-related.
Details on embryotoxic / teratogenic effects:
Laparotomy revealed no dead fetuses or runts at any tested dose level (100, 300 or 1000 mg Urea/kg bw/day).
No malformations were noted during external / internal macroscopic inspection at laparotomy, skeletal examination (according to DAWSON) or soft tissue examination (according to WILSON).
No test-item related variations were noted in the fetuses during external / internal macroscopic inspection at laparotomy or soft tissue examination (according to WILSON) at any tested dose level. Skeletal examination (according to DAWSON) revealed no test item-related variations or retardations.
All further prenatal changes noted are without any biological relevance.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table1. Reproductive data of the dams



Parameter

Group 1

Control

(n=20)

Group 2

100 mg/kg

(n=20)

Group 3

300 mg/kg

(n=20)

Group 4

1000 mg/kg

(n=20)

Corpora lutea

total

mean per dam

294

14.7

292

14.6

287

14.4

291

14.6

Implantation sites

total

mean per dam

285

14.3

290

14.5

281

14.1

289

14.5

Resorptions

total

mean per dam

14

0.7

10

0.5

5

0.3

18

0.9

Early resorptions

total

mean per dam

13

0.7

9

0.5

3

0.2

17

0.9

Late resorptions

total

mean per dam

1

0.1

1

0.1

2

0.1

1

0.1

Live fetuses

total

mean per dam

271

13.6

280

14.0

276

13.8

271

13.6

Dead fetuses

total

0

0

0

0

Pre-implantation loss [%]

per group ##1

mean per dam

3.1

2.9

0.7

0.6

2.1

2.0

0.7

0.9

Post-implantation loss [%]

per group ##2

mean per dam

4.9

5.1

3.4

3.3

1.8

1.6

6.2

6.2

Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.

##1: The statistical comparison of the pre-implantation loss per group was done by comparing the values of implantation sites/corpora lutea of the test group with the ratio of implantation sites/corpora lutea of the control group using the Chi2test (*/**: p ≤ 0.05/p ≤ 0.01).

##2: The statistical comparison of the post-implantation loss per group was done by comparing the values of live fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group using the Chi2test (*/**: p ≤ 0.05/p ≤ 0.01).

Applicant's summary and conclusion

Conclusions:
In this prenatal developmental toxicity study, the test item Urea was administered orally to female rats at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 1000 mg Urea/kg bw/day for the dams.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was also above 1000 mg Urea/kg bw/day.
No test item-related malformation was noted at any of the test item treated groups. There was no test item-related increase in the incidence of fetal external / internal, skeletal or soft tissue variations or skeletal retardations at any tested dose level.
In conclusion, no embryotoxic properties of the test item were noted during external / internal, skeletal and soft tissue examination up to the highest tested dose of 1000 mg Urea/kg bw/day by oral administration. Under the conditions of the study, Urea did not show any teratogenic potential.
Executive summary:

In this prenatal developmental toxicity study, the test item Urea was administered orally to female  pregnant CD® / Crl:CD(SD) rats by gavage at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy. Tap water was used as vehicle and the administration volume was 5 mL/kg bw/day. The measured actual concentrations of the test item in the test item vehicle mixtures were between 101.0% and 105.8% of the nominal concentrations, indicating correctly prepared formulations. On gestation day 21, the dams were laparotomised and examined for implantation sites, resorptions in the uterus and for the condition of the fetuses.On gestation day 21, the dams were laparotomised and examined for implantation sites, resorptions in the uterus and for the condition of the fetuses.

Examination of the dams:

No premature death was noted in the control group and in the treatment groups. No changes were noted in behaviour, the external appearance or the faeces.

No test item-related differences in body weight, body weight gain and food consumption were noted between the control group and the treatment groups.

The visual appraisal of the drinking water consumption revealed no test item-related influence at any tested dose level.

No test item-related changes were noted during the macroscopic inspection nor for the gravid uterus or carcass weights of the dams at necropsy.

No test item-related influence was noted on the reproductive parameters (number of implantation sites, resorptions and fetuses).

Examination of the fetuses:

No dead fetuses were noted in any of the test groups. No test item-related influence was detected on the prenatal fetal development with respect to the number of resorptions, number of live fetuses, fetal and placental weights, the values calculated for the post-implantation loss and the sex distribution of fetuses.

No malformations nor variations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.

Conclusions
The no-observed-adverse-effect level (NOAEL) was above 1000 mg Urea/kg bw/day for the dams.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was also above 1000 mg Urea/kg bw/day.

No embryotoxic properties of the test item were noted during external / internal, skeletal and soft tissue examination up to the highest tested dose of 1000 mg Urea/kg bw/day by oral administration. Under the conditions of the study, Urea did not show any teratogenic potential.