N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This Annex IX vertebrate study was conducted for other regulatory purposes required by the non-EU manufacturer appointing our company as Only Representative and therefore no testing proposal was submitted to ECHA before commencement of the study. The study results are relevant for the purposes of fulfilling the information requirements under REACH for this endpoint.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

EC Number:

876-151-9

Cas Number:

2292123-68-1

Molecular formula:

C27H24N2O7S2

IUPAC Name:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crl: CD(SD)

Details on species / strain selection:

This is strain is widely used in general toxicity studies and historical control data is available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Chares River Laboratories, Japan
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 5 weeks
- Weight at study initiation: 150.3 - 174.1g (males); 128.2-147.0g (females)
- Fasting period before study: not specified
- Housing: Stainless steel hanging cages, wire-mesh floors
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
Acclimation period: 5 days on receipt of animal at test facility

DETAILS OF FOOD AND WATER QUALITY:
- Pelleted diet
- Drinking water from city supply with chlorine level maintained at 3-5ppm. Water analysed twice per year in accordance with national regulations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

As required by the guideline

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed and mixed with corn oil to be suspended. The suspension was filled up with corn oil and treated with a homogeniser to prepare a 20.0 w/v% test item formulation (suspension). A part of the 20.0 w/v% formulation was taken while being stirred by a magnetic stirrer and diluted with corn oil to prepare 5.00 and 1.25 w/v% formulations (suspensions) - see Table 1 below.

On each preparation day, the formulation and vehicle for dosing were subdivided into plastic containers and stored at a cold place (refrigerator 1-10°C). The formulations were used within the
validated stability period. The containers of the formulations and vehicle were carried to the animal room at room temperature and dosed.

VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle for general toxicity studies
- Concentration in vehicle: 1.25, 5 and 20% w/v of test item

Analytical verification of doses or concentrations:

yes

Remarks:

High Performance Liquid Chromatography (HPLC)

Details on analytical verification of doses or concentrations:

The stabilities of the 20.0 and 0.200 w/v% test substance formulations measured after storage for 13 days. The concentrations measured were within 100 +/-10% of the
measured concentrations immediately after preparation. Therefore the substance was determined to be stable in the vehicle for at least 12 days after preparation.

For homogenicity, the coefficients of variation (CV) of the measured test item concentrations in the upper, middle and lower layers of the 20 and 0.200 w/v% test item formulations were confirmed within 5%.

Duration of treatment / exposure:

90 days (males), 91 days (females) (plus 28 days for recovery group)

Frequency of treatment:

Daily dosing by gavage

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Mid-dose

Doses / concentrations 3

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

Low dose

No. of animals per sex per dose:

10 male, 10 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on 28-day repeated-dose toxicity study. No toxicity observed at any dose. As the study is longer in duration the low and mid doses were set at lower concentrations. Highest dose was maintained at 1000 mg/kg/day for this study.
- Rationale for animal assignment (if not random): Body weight-stratified randomisation.
- Fasting period before blood sampling for clinical biochemistry: Overnight (16 - 20 hours)
- Rationale for selecting satellite groups: same as all test item groups and vehicle controls
- Post-exposure recovery period in satellite groups: 28 days
- Dose range finding studies: existing 28-day repeated dose study data used for setting dose range. No specific DRF performed.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during the dosing period, the animals were observed before and after dosing. For the recovery period, the animals were observed once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before dosing period and once per week thereafter. After initiation of dosing, the animals were examined in a blind test basis, with random numbering and observation labels without identifying the dose levels.
Parameters in Table 2 were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 90 of the dosing period. In the recovery group, in addition to the above, Days 1, 7, 14, 21 and 28 of the recovery period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the last week of the dosing period
- Dose groups that were examined: High dose group only

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day after dosing period, day after recovery period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 100
- Parameters checked in Table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day after dosing period, day after recovery period.
- Animals fasted: Yes
- How many animals: 100
- Parameters checked in Table 4 were examined.

PLASMA/SERUM: Yes
- Time of blood sample collection: Day after dosing period, day after recovery period.
- Animals fasted: Yes
- How many animals: 100

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No - omitted as no abnormalities were observed in the 28-day repeated dose toxicity study

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 5 for organs and tissues collected)
Performed on the day after the dosing period for the main groups and the day after the recovery period for the recover group. External surfaces of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities and their contents were observed. For females, vaginal smears were collected before gross necropsy, stages of the oestrous cycle were determined with light microscope after Giemsa staining.

HISTOPATHOLOGY: Yes (see table 6)

Statistics:

Body weights and food consumption during the dosing period, and parameters of the haematological and blood chemical examinations, organ weights and body weights on the necropsy day for the main group were analysed by the Bartlett's test for homogeneity of variances. When the variances were homogeneous at a significance level of 5% in this analysis, the Dunnett's test was performed. When variances were not homogeneous, the nonparametric Dunnett's test was performed. Nonparametric Dunnett's test used for the analysis of the frequencies of defecation and urination during the dosing period.

Body weights and food consumption during the recovery period, and parameters of the haematological and blood chemical examinations, organ weights and body weights on the necropsy day for the recovery groups were analysed by the F-test for variance ratio. When there were no significance differences at a significance level of 5% in this analysis, the Student's t-test was performed. When there were significant differences, the Aspin-Welch t-test was performed. The Mann-Whitney U-test was performed.
The statistical analysis was two-sided and was compared with the control group for the treatment groups. Significance levels set at 1 and 5%

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):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
In males, a significant increase of red blood cell count was observed in the 1000 mg/kg group. In the 62.5 mg/kg group, a significant prolongation of prothrombin time was observed although it was not dose-dependent. No significant changes were observed in the 250 mg/kg groups.

In females, a significant decrease of reticulocyte count ratio was observed in the 250 mg/kg group although it was not dose-dependent. No significant changes were observed in the 62.5 or 1000 mg/kg groups.

Recovery groups: In either sex, no significant changes were observed in the 1000 mg/kg group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
In males significant decreases were observed in aspartate aminotransferase (AST) of the 62.5 and 250 mg/kg and y-glutamyl transpeptidase of the 62.5 mg/kg group, although they were not dose-dependent. No significant changes were observed in the 1000 mg/kg group.
In females, a significant decrease of thyroxin was observed in the 1000 mg/kg group. A significant increase in triiodothyronine in the 250 mg/kg group and significant decrease of the thyroid stimulating hormone in the 62.5 and 250 mg/kg groups were observed, although these were not dose-dependent.

Recovery groups: In males, significant increases were observed in AST, alanine aminotransferase, HDL-cholesterol, blood urea nitrogen, creatinine, total protein, albumin, glucose, inorganic phosphorus and calcium of the 1000 mg/kg group. In females, no significant changes were observed in the 1000 mg/kg group.

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
In males, a decrease in size of left lobe of the thyroid was observed in 1/10 animals of the 1000 mg/kg group. No abnormalities were observed in the 62.5 or 250 mg/kg groups or control groups.
In females, blackish region of mucosa of the glandular stomach was observed in 1/10 animals of the 250 mg/kg group. Cyst of the pituitary gland was observed in 1/10 animals of the 62.5 mg/kg group. Nodule of the vagina was observed in 1/10 animals of the control group. No abnormalities were observed in the 1000 mg/kg group.

Recovery groups:
In males, mass of the jejunum and whitish substance in the abdominal cavity were observed in 1/5 animals of the 1000 mg/kg group. No abnormalities were observed in the control group.
In females, no abnormalities were observed in the control group or 1000 mg/kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In either sex, no findings considered as treatment-related changes were observed. In males, the following findings were observed and considered as spontaneous lesion:
- Focal inflammation in the lung in 1/10 animals of the 1000 mg/kg group
- Mineralisation in Peyer's patches in the jejunum in 1/10 animals of the control group
- Focal fibrosis in the heart in 1/10 animals of the 1000 mg/kg group
- Focal myocarditis in the heart in 2/10 animals of the 1000 mg/kg group and 5/10 animals of the control group.
- Basophilic tubules in the kidney in 1/10 animals of the 1000 mg/kg group
- Increased eosinophilic bodies in the kidney in 1/10 animals of the control group
- Solitary cyst in medulla in the kidney in 1/10 animals of the control group
- Lymphocyte infiltration in the ventral prostate in 5/10 animals of the 1000 mg/kg group and 3/10 animals of the control group
- Focal myositis in the skeletal muscle in 1/10 animals of the 1000 mg/kg group

In females, the following findings were observed and considered as the spontaneous lesion:
- Erosion of fundic mucosa in the glandular stomach in 1 animal of the 250 mg/kg group in which blackish region of mucosa of the glandular stomach was observed in the gross necropsy
- Mineralisation in Peyer's patches in the jejunum in 1/10 animals of the 1000 mg/kg group
- Microgranuloma in the liver in 1/10 animals of the 1000 mg/kg group and 1/10 animals of the control group
- Mineralisation in cortico-medullary junction in the kidney in /10 animals of the the 1000 mg/kg group and 2/10 animals of the control group
- Solitary cyst in medulla in the kidney in 1/10 animals of the 1000 mg/kg group
- Squamous epithelial cyst in the vagina in 1/10 animals of the control group in which nodule of the vagina was observed in the gross necropsy
- Cyst formation in pars intermedia in the pituitary gland in 1 animal of the 62.5 mg/kg group in which the cyst of the pituitary gland was observed in the gross necropsy
- Rathke's pouch remnant in pituitary gland in 1/10 animals of the 1000 mg/kg group
- Focal myositis in the skeletal muscle in 1/10 animals of the control group

Recovery groups:
In males, the following findings were observed and considered as the spontaneous lesion
- Ectopic jejunal tissue in the serosa of jejunum in 1 animal of the 1000 mg/kg group in which mass of the jejunum and whitish substance in the abdominal cavity were observed in the gross necropsy
In females, no abnormalities were observed in the thyroid in control or 1000 mg/kg groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Summary result tables are provided as an attachment to this endpoint study record.

No treatment-related effects were observed by 90 or 91 day repeated doses of the test material.

In the haematological examinations, an increase of the red blood cell count was observed in males of the 1000 mg/kg group.  However, the increase was lsight change (106% compared with the control group) and corresponding changes were not observed in the gross necropsy or histopathological examination.  Therefore, the change was considered to be incidental.

In the clinical chemistry examinations, a decrease of thyroxin was observed in females of the females of the 1000 mg/kg group.  However, no abnormalities were observed in triiodothyronine or thyroid stimulating hormone and corresponding changes were not observed in the gross necropsy or histopathological examination.  Therefore, the change was considered to be incidental.

Other changes observed at the end of the dosing period were not dose-dependent changes, changes in the recovery group only, no other related changes, or occasional findings a spontaneous lesions.  Therefore, these changes were considered to be incidental changes not related to the administration of the test substance.

 

Applicant's summary and conclusion

Conclusions:

The No-Observed-Adverse-Effect Level (NOAEL) of the test substance under the condition of this study was considered to be 1000 mg/kg/day since no adverse effects were observed/detected in either sex of the 1000 mg/kg groups.