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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-08-19 to 2014-12-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

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)
Version / remarks:
22 March 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2-benzenesulfonamidophenyl)-3-phenylurea
EC Number:
806-543-7
Cas Number:
215917-77-4
Molecular formula:
C19H17N3O3S
IUPAC Name:
1-(2-benzenesulfonamidophenyl)-3-phenylurea
Test material form:
solid: particulate/powder

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 Japan, Inc. (Hino Breeding Centre).
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) Males: 320.9-367.7 g; Females: 214.9-240.8 g
- Housing: Males and females (except during the gestation and lactation periods); stainless steel cages. For females during the gestation and lactation periods; polymethylpentane cages.
- Diet: ad libitum (except during the urine collection and during measurement of motor activity) autoclaved sterilized pellet diet (CRF-1, oriental yeast co. Ltd.). Animals were fasted on the day before scheduled necropsy.
- Water: ad libitum, except during the measurement of motor activity
- Acclimation period: 6 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 24.3
- Humidity (%): 48.6 to 69.8
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% w/v% methyl cellulose aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing preparations were prepared once every 4-8 days (based on the results of previous stability and homogeniety assessments). The test substance was initially ground in an agate pestle and mortar, and a few drops of vehicle added. This suspension was then transferred into a measuring cylinder. The appropriate volumes of the vehicle was added to the measuring cyclinder and mixed to achieve the required concentrations which were then added into brown glass vials and stored refrigerated until use.

VEHICLE
- Concentration in vehicle: 0, 11, 33 and 100 mg/mL
- Amount of vehicle: Dose volume of 10 mL/kg bw/day
- Lot/batch no.: PDG1792
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of mating was apparent (upto 6 days).
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of gestation.
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the initial test substance preparation, 15 mL of each of the analytical samples were tested for homogeneity and analytical determination. The measured concentrations were within acceptable ranges (actual values 98.4 to 100.3% of nominal).
Duration of treatment / exposure:
Males: From 14 days before mating (Days 1 to 15) until the day before the necropsy through the mating period (42 days in total).
Females: From 14 days before mating (Days 1 to 15) until Day 4 of lactation (day of delivery was Day 0 of lactation) through the mating and pregnancy periods and delivery. One female not which did not deliver was kept until day before the necropsy.
Frequency of treatment:
Parental males and females daily by oral gavage.
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 11 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
110 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- A total of 106 rats were used on study (48 males and 58 females).
- Each test group consisted of 12 males (including 5 males for the control and 1000 mg/kg bw/day groups in the recovery test) and 12 females (5 females
each were added for the control and 1000 mg/kg bw/day groups in the recovery test).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A preceeding 14 Day repeated dose oral study with the test substance to rats, indicated that at a high dose of 1000 mg/kg bw/day would be expected to provide obvious toxicological changes.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
See Chapter 7.5.1, Supporting study
Oestrous cyclicity (parental animals):
Vaginal smears were collected with swabs from all test females in the morning (same time every day) from the initial dosing day to the day of mating or end of the mating period to confirm the estrous cycle. The obtained smears were collected on a plate for each animal, and stained with Giemsa.The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E), and metestrus (M). The mean estrous cycle (number of
days from the estrous period to the next estrous period) and the number of the estrous period during the test period were calculated.
Sperm parameters (parental animals):
Not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities.
Postmortem examinations (parental animals):
Necropsy and histopathology procedures are described in Chapter 7.5.1, Supporting study
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on Day 4 of lactation.
- These animals were subjected to postmortem macroscopic examination (external anomalies).

GROSS NECROPSY
- Gross necropsy consisted of a check for external anomalies, including that in the oral cavity.

Statistics:
Histopathological findings were analysed using Fisher's exact probability test. Copulation index, fertility index, gestation index, delivery index and sex ratio were analysed using the Chi-squared test. Implantation index, stillborn rate, external anomaly index, external anomaly typing index, live birth index, and viability index were analysed using Wilcoxon's rank sum test.

Number of rearing, grip strength, motor activity, body weights, food consumption, urinalysis (reagent strip), hematology, blood chemistry, absolute and relative organ weight, estrous cycle, number of estrus, days until copulation, gestation length, number of corpora lutea, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring (both sexes) were initially analysed using Bartlett's test. When the data was homogenous the Dunnet's multiple comparison test was applied, and when not homogenous, Steel's multiple comparison test was applied for the control group and each test group. For the urinalysis reagent strip, Steel's test was performed.
Reproductive indices:
Compulation index (%), fertility index (%), gestation length, implantation index (%), gestation index (%) and delivery index (%) were the determined reproductive indices.
Offspring viability indices:
Live birth index (%), stillborn rate (%), Viability index on Day 4 (%), Sex ration (%), external anomaly index (%), and external anomaly typing index (%), were the determined offspring viability indices.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal clinical signs were observed in either sex of any group throughout the
dosing or recovery period, including the gestation and lactation periods of pregnant
females.
Mortality:
no mortality observed
Description (incidence):
No death occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant difference was observed in any sex in the treated-groups as compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistically significant difference was observed in any sex in the treated-groups as compared to the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):

No treatment-related changes were observed in any treated group.
A low value of eosinophil ratio was observed in males of the 110 mg/kg bw/day group and above; however, as no significant change was noted in eosinophil count or leukocyte count and no related changes were observed in any examination, it was not considered to be toxicologically significant. A high value of reticulocyte ratio was observed in males of the 330 mg/kg bw/day group; however, the change was not considered to be treatment-related because there was no dose-dependency. No statistically significant difference was observed in females of any treated-group.
At the end of recovery period: A low value of white blood cell count was observed in satellite females of the 1000 mg/kg bw/day group, however, it was judged to be toxicologically insignificant, as this was no observed at the end of the dosing period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed in any treated group.
A high value of inorganic phosphorus was observed in males of the 1000 mg/kg bw/day group. However, it was judged to be toxicologically insignificant, as no related changes were observed in the other blood chemistry items and were observed in any examinations. A low value of sodium was observed in males of the 110 and 1000 mg/kg bw/day groups. However, it was judged to be toxicologically insignificant, as the change was noted in only a value that was slightly higher than the historical data in the test facility (min-max: 143.6 to 144.7 mmol/L; 2010 to 2013), and no related changes were observed in any examinations. A low value of albumin and a high value of creatinine were observed in females of the 330 mg/kg bw/day group; however, these were not considered to be treatment-related because there was no dose-dependency.
At the end of recovery period: A high value of ASAT was observed in males of the 1000 mg/kg bw/day group, and a low value of Ca and a high value of Na were observed in females of the 1000 mg/kg bw/day group. However, the changes were judged to be toxicologically insignificant, as these were not observed at the end of the dosing period.
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
In the qualitative analysis, no statistically significant difference was observed in males in
the treated-groups as compared to the control group.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The various histopathological changes were observed in the animals of the control and 1000 mg/kg bw/day groups subjected to necropsy at the end of the treatment. However, these changes were judged to be not related to the test substance treatment, as such changes are nonspecifically observed in rats, and there was no apparent dose-dependency in their incidences. No histopathological change was observed in the ovary of the non-pregnant animal.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes in estrous cycle were observed in females of any treated group. One female of the 1000 mg/kg bw/day group showed continuous estrus for 4 days (from Day 5 to Day 8) and extension of proestrus for 5 days (from Day 10 to Day 14), suggesting an irregular estrous cycle. In one female of the 1000 mg/kg bw/day group, irregular estrous cycle (6-day cycle) was observed. However, no statistically significant change was observed in the mean estrous cycles or estrus count between the control group and any treated group.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related differences in:
- Estrus cycling
- precoital time
- the number of pregnant females
- duration of gestation
- mean numbers of corpora lutae
- number of total, pre-implantation and post-implantation sites

Selected results by dose level (0, 110, 330 or 1000 mg/kg bw/day).
- females mated: 12/12, 12/12, 12/12, 12/12
- females pregnant: 12/12, 12/12, 12/12, 11/12
- females with live pups: 12/12, 12/12, 12/12, 11/12
- Copulation indices (%): 100, 100, 100, 100
- fertility Indices (%): 100, 100, 100, 91.7

Effect levels (P0)

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: No adverse effects at highest dose tested

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant difference was observed between the control group and any treated group in body weight on days 0 and 4 of postpartum.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No external abnormalities were observed in the offspring on Day 4 of lactation in any group.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No statistically significant difference was noted in number of live offspring at birth, number of stillborn at birth, sex ratio, stillborn rate, or viability indexon Day 4 between the control group and any treated group. In addition, a high value of the birth index was observed in the 1000 mg/kg group; however, this change was judged to be not related to the test substance treatment because the post-implantation losses were a few.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects at highest dose tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
There was no evidence of adverse effects on parental reproductive indices and litter data following repeated oral exposure of the test substance, with the NOAEL set as 1000 mg/kg bw/day in parental animals and offspring under the conditions of the test.
Executive summary:

A study according OECD TG 422 was performed. The substance was administered repeatedly by oral gavage at dose levels of 0 (control group), 110, 330, and 1000 mg/kg bw/day from 14 days before mating through mating for 42 days in males and satellite females, and 14 days before mating through gestation and parturition until Day 4 of lactation in females to determine the repeated dose toxicity and reproductive and developmental toxicity, as well as reversibility of the changes observed. Each test group consisted of 12 males (including 5 males for the control and 1000 mg/kg bw/day groups in the recovery test) and 12 females (5 females each were added for the control and 1000 mg/kg groups in the recovery test). The control group received the vehicle (0.5 w/v% methylcellulose aqueous solution) only.


No death occurred during the study. No changes related to the test substance treatment were observed in the clinical signs, functional observational battery, body weight, food consumption, hematological examination, blood chemical examination, necropsy, organ weight or histopathological examination in males or females or urinalysis in males.


In the parental animals, no changes related to the test substance treatment were observed in the estrous cycle, copulation index, fertility index, gestation index, gestation length, number of corpora lutea or implantation sites, implantation index, delivery index, nor in the parturition or maternal behavior. In the offspring, no changes related to the test substance treatment were observed in the sex ratio, birth index, or viability index on day 4 of postpartum, nor in the external examination or body weight.


The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg bw/day in both sexes under the conditions of this study. The NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day in parental animals and offspring under the conditions of this study.