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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Lot no.: 140220
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 (e.g. ad libitum): 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 (e.g. ad libitum): Ad libitum (except during the measurement of motor activity) well water.
- 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
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 (if gavage): Dose volume of 10 mL/kg/day
- Lot/batch no. (if required): 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
Remarks:
Doses / Concentrations:
0, 110, 330 and 1000 mg/kg/day.
Basis:
nominal conc.
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 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).
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/day would be expected to provide obvious toxicological changes.
Parental animals: Observations and examinations:
See Chapter 7.5.1, record Repeated dose toxicity: oral.001
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, record Repeated dose toxicity: oral.001
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.
Clinical signs:
not examined
Description (incidence and severity):
Examined in section 7.5.1
Body weight and weight changes:
not examined
Description (incidence and severity):
Bodyweight examined in section 7.5.1
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Bodyweight examined in section 7.5.1
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Examined in section 7.5.1
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Reproductive performance
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

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No adverse effects at highest dose tested
Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
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.

BODY WEIGHT (OFFSPRING)
No statistically significant difference was observed between the control group and any treated group in body weight on days 0 and 4 of postpartum.

GROSS PATHOLOGY (OFFSPRING)
No external anomalies were observed in any offspring.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No adverse effects at highest dose tested
Key result
Reproductive effects observed:
not specified
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/day in parental animals and offspring under the conditions of the test.

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)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-{2-[(phenylcarbamoyl)amino]phenyl}benzenesulfonamide
EC Number:
806-543-7
Cas Number:
215917-77-4
Molecular formula:
C19H17N3O3S
IUPAC Name:
N-{2-[(phenylcarbamoyl)amino]phenyl}benzenesulfonamide
Specific details on test material used for the study:
Lot no.: 140220

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 (e.g. ad libitum): 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 (e.g. ad libitum): Ad libitum (except during the measurement of motor activity) well water.
- 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 oral 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 (if gavage): Dose volume of 10 mL/kg/day
- Lot/batch no. (if required): PDG1792
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 which did not deliver was kept until day before the necropsy.
Frequency of treatment:
Parental males and females daily by oral gavage.
Doses / concentrations
Remarks:
0, 110, 330 and 1000 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 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).
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/day would be expected to provide any obvious toxicological changes.

- Post-exposure recovery period in satellite groups: Yes in males and females.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before and after dosing) during the dosing period, and once a day in the other periods.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical observations were performed once a day before commencement of dosing, then once a week during the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: The test and recovery males were weighed on Days 1, 8, 15, 22, 29, 36, and 42. The recovery males were also weighed on Days 43, 50, and 56. The satellite females were weighed on the same manner as the recovery males. The test females were weighed on
Days 1, 8, and 15, Days 0, 7, 14, and 20 of gestation, and Days 0 and 4 of lactation. The final body weight was also measured on the day of the scheduled necropsy.

FOOD CONSUMPTION: YES
- Food consumption was measured for Days 1 to 8, 8 to 15, 29 to 36, and 36 to 40 in test and recovery males, and for Days 43 to 50 and 50 to 54 in recovery males only. For females, it was measured fro Days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 42, 43 to 50 and 50 to 56 in satellite females.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 43 for test males, and Day 57 for the recovery males and satellite females, and Day 5 of lactation for the test females.
- Anaesthetic used for blood collection: Yes; intraperitoneal injection of sodium pentabarbitol (30 mg/kg).
- Animals fasted: Yes; for 19-23 hours before sampling.
- How many animals: 5 animals for the test males, all recovery males and satellite females, and 5 females from the earlier partuition date.
- Parameters examined: Leukocyte, RBC, HGB, Haematocrit, MCV, MCH, MCHC, reticulocyte ratio and count, differential leukocyte ratio/count, PT and APTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Straight after sampling for haematology.
- Animals fasted: Yes; for 19-23 hours before sampling
- How many animals: 5 animals for the test males, all recovery males and satellite females, and 5 females from the earlier partuition date
- Parameters examined: Total protein, albumin, A/G ratio), ASAT, ALAT, GGT, ALP, total bilirubin, total bile acids, total cholesterol, triglycerides, glucose, urea nitrogen, creatinine, calcium, inorganic phosphorus, sodium, potassium and chloride.

URINALYSIS: Yes (In males)
- Time schedule for collection of urine: 5 males in the final dosing week (week 40).
- Animals fasted: Yes (water was available).
- Parameters examined: pH, protein, glucose, ketone and occult blood all analysed using a reagent strip. As no abnormalities related to treatment were detected in any of the parameters analysed, the urinary sediment and accumulated urine test and the urinalysis during the recovery period was not conducted.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The functional test and motor activity measurements were observed in 5 males per group once in Week 6. For females, 5 dams from the nearer parturition date were selected from each group and observed once during the lactation period. The functional test and motor activity observations were not measured during the recovery periods, as no treatment related changes were noted during the dosing periods.
- Dose groups that were examined: All
- Battery of functions tested: sensory activity to stimuli / grip strength / motor activity.
Sacrifice and pathology:
All animals were sacrificed by exsanguination under anaeshesia and subjected to necropsy (after sampling for haematology and blood chemistry investigations).
Samples of the following tissues/organs were preserved in 10% phosphate buffered formalin (testes and epididymides in Bouin's fixative and the eyes in a davidson's solution):
- Brain (including cerebrum, cerebellum, medulla oblongata and pons).*
- Pituitary
- Spinal cord (cervical)
- Eyes
- Submandibular lymph node
- Submandibular gland
- Thyroid*
- Parathryoid
- Thymus*
- Trachea
- Lungs (including bronchus)
- Heart*
- Stomach
- Duodenum
- Jujunum
- Ileum (including payer's patch)
- Cecum
- Colon
- Rectum
- Mesenteric lymph node
- Liver*
- Pancreas
- Spleen*
- Kidney*
- Adrenal*
- Urinary bladder
- Testis*
- Epididmymis*
- Seminal vesicle (including dorsolateral and coagulating gland)*
- Prostate (ventral)*
- Ovary*
- Uterus
- vagina
- Femur
- bone marrow (femur)
- Sternum
- Bone marrow (sternum)
- M.Biceps femoris
- sciatic nerve
- Other gross legions
Tissues marked (*) were also weighed

Histopathology
Tissues from 5 high dose and control animals were processed and stained with haematoxylin and eosin prior to microscopic examination. One non-pregnant female also had the ovaries stained an examined. As no test substance related changes were observed at 1000 mg/kg, the additional microscopic examination of the intermediate groups was not performed.
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
In males only
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No deaths occured during study and no abnormal clinical signs were observed throughout the dosing and recovery periods.

BODY WEIGHT AND WEIGHT GAIN: No statistically significant differences were observed in any sex in the treatment-groups compared with the controls.

FOOD CONSUMPTION: No statistically significant differences were observed in any sex in the treatment-groups compared with the controls.

HAEMATOLOGY: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods. Any effects observed were deemed to be non-dose dependant in their incidence and changes were not seen at the end of the dosing period.

CLINICAL CHEMISTRY: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods. Any effects observed were deemed to be non-dose dependant in their incidence, changes were not seen at the end of the dosing period, or values were similar to historical control values.

URINALYSIS: In the qualitative analysis, no statistically significant difference was observed in males in the treated-groups as compared to the control group.

NEUROBEHAVIOUR: There were considered not to be any treatment related changes with regards to reactivity to stimuli, grip strength and motor activity in males or females at any treatment group. No treatment related changes were also observed in the detailed clinical investigations (hand-held or open field observation). Changes observed during the motor activity investigations, were not considered treatment related as no dose-repsonse was observed.

ORGAN WEIGHTS: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods. Any effects observed were deemed to be non-dose dependant in their incidence, changes were not seen at the end of the dosing period and/or there was no associated histopathological findings.

GROSS PATHOLOGY: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods, including the non-pregnant female. Any effects observed were deemed to be non-dose dependant in their incidence.

HISTOPATHOLOGY: NON-NEOPLASTIC: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods, including the non-pregnant female. Any effects observed were deemed to be non-dose dependant in their incidence.

Effect levels

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

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg/day in both sexes under the conditions of this study, as there were no effects of parameters associated with repeated dose toxicity.