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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:
2021-05-11 to 2021-06-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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 animals

Species:
rat
Strain:
Wistar
Remarks:
Han: WIST
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90., 1103 Budapest, Hungary
- Age at study initiation: ca. 10 weeks (females), 33-34 weeks (males for mating)
- Weight at study initiation: The group averages of the body weight of the females were as similar as possible on the first day of gestation (182-232 g)
- Fasting period before study: no
- Housing:
Before mating: 1-2 females per cage, 2 males per cage
During mating: 1 male and 1-2 females / cage
During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum, ssniff® SM Rat/Mouse-Zucht+Haltung" Autoclavable
complete feed for rats and mice – breeding " produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: 6 days for females

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 24.6
- Humidity (%): 44 - 61
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Methylcellulose (1 %)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (Methylcellulose (1 %)) at concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand for not longer than 2 days and stored in a refrigerator (5±3 °C) until use. A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals.
VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water. Therefore, Methylcellulose (1 %) was used for preparing formulations appropriate for oral administration. Methylcellulose (1 %) is a suitable vehicle for the test item
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean concentration of formulation samples was in the range of 106-109 % compared to the corresponding nominal concentration (within the acceptance criteria). The formulations were considered as homogenous since the difference of the measured concentrations of parallel samples was not more than 15% (1.2-3.3%).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one to two females
- Length of cohabitation: 2 - 4 hours
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19.
Frequency of treatment:
daily
Duration of test:
15 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
26
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of the dose range finding study, where oral treatment of pregnant Han: WIST rats with the dose levels of 1000, 300 and 100 mg/kg bw/day of NKK-1304 revealed neither maternal nor fetal toxicity.
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: in the morning on the day of necropsy within a short timeframe (not exceeding two hours).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for behavior and general condition; twice daily for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the female rats was measured at least once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g). The corrected body weight was calculated for the 20th day of pregnancy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Thyroid glands together with the parathyroid glands of all sperm positive females were weighed and recorded with a precision of 0.001 g after fixation in 4 % (v/v) buffered formaldehyde solution.
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
Blood sampling:
- Plasma: No
- Serum: Yes
- Volume collected: least 1.0 mL blood
-Other: Serum samples were divided into two aliquots (one for FT3 and FT4 as well as one for TSH level determination) and stored between -15 to -30 °C
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: The anogenital distance (AGD) of each fetus was determined (accuracy 1 mm).
Statistics:
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test. Chi2 test was performed if feasible.
Indices:
- Pre-implantation loss
- Post-implantation loss
- Sex distribution
- External abnormalities/litter
- Visceral abnormalities/litter
- Skeletal abnormalities/litter
Historical control data:
Yes, provided by test laboratory.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female in the 100 mg/kg bw/day group was moribund and euthanized in the course of the study on g.d. 11. This female had clinical signs from g.d. 9 such as hunched back, piloerection and reduced activity as well it was lying on the side and had dyspoea on g.d. 11. Considering that none of the other animals died, was moribund or had clinical signs in the experimental groups, this was judged as unrelated to the test item.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight/gain (including corrected body weight/gain) values were similar in the groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no reductions in the food consumption observed. In the treatment period, there were no statistically significant differences indicated. Before the treatment period, from g.d. 3 to 5, a slightly but statistically significantly higher (p<0.05) mean food consumption was seen in the 300 and 1000 mg/kg bw/day which was considered as biologically not relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no statistically significant differences observed in the measured TSH, FT3 and FT4 hormone levels in the control, 100, 300 and 1000 mg/kg bw/day groups.
Clinical biochemistry findings:
not examined
Endocrine findings:
no effects observed
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):
The mean thyroid weight (including relative values) was statistically significantly lower (0.0265 g, p<0.05) in the 1000 mg/kg bw/day dose group. However, according to the historical control database, the value was above the historical control range (0.0219-0.0224 g). Considering this, and that there were no differences in the thyroid and TSH hormone levels indicated, as well as there were no lesions found in the thyroids at histopathology, it was not considered as treatment-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dilated renal pelvis occurred with low incidences and without a dose response in one control female. Therefore, these were considered as unrelated to the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
According to the expert’s evaluation, there were no test item- related lesions observed upon histological examinations of the thyroid tissue in all treatment groups compared to the control. There were no other organs examined since no lesions were detected in the treatment groups during necropsy.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There was no increase indicated in the mean of pre- and post-implantation loss. According to the Chi square evaluation, the number of pre-implantation loss was statistically significantly lower in the 100 mg/kg bw/day dose group and statistically significantly higher in the 300 mg/kg bw/day dose group without a dose response, hence not considered as treatment related.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects 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: highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no differences in the body weight of the fetuses.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The number of male fetuses was statistically significantly higher, and the number of female fetuses statistically significantly lower in the 300 mg/kg bw/day group versus control. This was considered as not relevant, since the sex distribution was near to 50-50%.
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of litters with malformed fetuses was 2/23, 1/22, 1/23 and 1/23 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively. There were no statistically significant differences in the incidence of malformations over all dosing groups.
The number of evaluated fetuses was 240, 241, 238 and 233 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Variations: There were three pale fetuses found in the control and one with neck edema in the 100 mg/kg bw/day group.

Malformations: One fetus had umbilical hernia in the control group.

Growth retardation (small fetuses): There was no significant of dose related difference in the incidence of small fetuses.

Placentas, amniotic fluid: In one litter (in the same as with the three pale fetuses), the amniotic fluid was brownish and two of the placentas had clotted bloody margin in the control group.

Considering the low incidences at external examination, and lack of dose response, the findings were not attributed to an effect of the test item.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 121, 121, 119 and 115 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

There was no statistically significant increase observed in the skeletal abnormalities (variations and malformations).

Malformations:
There was one malformed fetus found in each group including control.

One fetus had vertebra agenesis (5 lumbar vertebrae) and a misshapen LI vertebra in the 300 mg/kg bw/day group. This was found in the same litter with the fetus with hydronephrosis.

One fetus had bent scapula in the control group. One fetus had also bent scapula and in addition bent radius, ulna, femur, tibia and fibula in the 100 mg/kg bw/day dose group.

One fetus had hyperflexion of the left forelimb in the 1000 mg/kg bw/day group.

Considering the low incidences or lack of dose response, the skeletal findings were not proved as treatment related.

Variations:
Delayed ossification of skull bones, sternebra, vertebrae, pelvic girdle, metacarpal/metatarsal, as well as slightly open palatine, misaligned or bipartite ossification of sternebra, wavy ribs or interrupted 14th rib, bipartite or dumb-bell shaped ossification of vertebral centra as well as slightly bent scapula were observed without statistically significant differences among the dose groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 119, 121, 119 and 118 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. There were no statistically significant increases in the incidence of visceral variations and malformations.

Variations:
Slightly dilated IIIrd ventricle was recorded in one fetus in the 300 mg/kg bw/day group.

Bilateral hydroureter was found in two fetuses in the 100 mg/kg bw/day group. Hydroureter accompanied with dilated renal pelvis was observed in one fetus in the control and one in the 100 mg/kg bw/day group.

Malformations
Marked hydroureter and in addition hydronephrosis was observed in one fetus in the 300 mg/kg bw/day group.

Considering the low incidences or lack of dose response, the findings at visceral examination were not judged as treatment related.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity including teratogenicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the observations the NOAELs from a OECD TG 414 study in rats were determined as follows:
NOAEL (maternal toxicity): 1000 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity):1000 mg/kg bw/day
Executive summary:

The substance was examined for its possible prenatal developmental toxicity in a study according OECD TG 414/GLP. Groups of 26 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle 1% aqueous methylcellulose. The treatment volume was 5 mL/kg bw.


A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The substance in 1% aqueous methylcellulose was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 20 and 250 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 106 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing.


During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses.


About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.


After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.


Results


In total, on gestation day 20 there were 23 evaluated litters in the control, 300 and 1000 mg/kg bw/day groups, respectively, as well as 22 in the 100 mg/kg bw/day group.


One non pregnant female was moribund in the course of the study in the 100 mg/kg bw/day group after clinical signs such as hunched back, piloerection and reduced activity, as well as dyspnoea and lying on the side due to an unknown reason. Necropsy revealed no macroscopic changes in the organs.


The other animals had no clinical signs and there were no treatment related pathological changes found.


The food consumption and body weight of the animals was similar in each group.


The mean of thyroid weight was statistically significantly lower in the 1000 mg/kg bw/day group (p<0.05), however the value was above the historical control level. Moreover, the treatment did not result in histological changes of the thyroid gland in any of the dose groups and the measured hormone levels were similar in the groups.


There were no treatment related differences observed in the pre- and post implantation loss. The number of implantations and viable fetuses as well as their sex distribution was similar in the groups.


The fetal weight was equal in the groups. There were no significant differences in the ano-genital distance and placental weight parameters. There were no significant differences in the incidence of body weight retarded (smaller) fetuses.


At fetal examinations, there were no significant differences in the incidence of malformations and variations during external, visceral and skeletal examinations at any dose level.


Conclusion


Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with the substance at the dose levels of 1000, 300 and 100 mg/kg bw/day caused no mortality, clinical signs and necropsy or histopathology changes of the maternal animals. The treatment with the test item had no impact the food consumption and body weight/corrected body weight/gain. Thyroid-related parameters (FT3, FT4 TSH hormone levels and histopathology) were not affected by treatment. Statistically significantly lower thyroid weight in the 1000 mg/kg bw/day dose group was not attributed to the treatment based on historical control data and lack of changes in the hormone levels or absence of histopathology lesions.  The test item caused no differences in the intrauterine parameters. No test item related differences in the incidence of malformations and variations as well as growth retardation or placental changes over all dosing groups were observed.


Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:


NOAEL (maternal toxicity):       1000 mg/kg bw/day


NOAEL (developmental toxicity


including teratogenicity):           1000 mg/kg bw/day