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

Toxicity to reproduction

Administrative data

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

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 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 29 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

In accordance with guideline

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:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

This strain is established as a laboratory animal and test facility has historical data of this strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) Males: 376-378 g; Females: 224-225 g
- Fasting period before study: not applicable
- Housing: Stainless steel cages with wire-mesh floors, 2-3 per cage for acclimation. After group allocation, males and females were housed individually. Enrichment provided. In the mating period, each female was housed in a male's cage in each dose group. Copulated females were housed in polycarbonate cages. Pups housed with dams after delivery.
- Diet (e.g. ad libitum): pelleted diet, ad libitum
- Water (e.g. ad libitum): Main water supply chlorinated, ad libitum
- Acclimation period: 14 days
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

Vehicle:

corn oil

Remarks:

Use as a vehicle for the substance in other toxicological studies

Details on 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).

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, 5.0 and 20% w/v of test item

Details on mating procedure:

- M/F ratio per cage: 1 to 1
- Length of cohabitation: until copulation or maximum of 14 days
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually in polycarbonate cages with enrichment

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 0.02 %w/v formulations were analysed using HPLC after preparation and after 13 days of storage in a cold place. 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 20.0 and 0.02 %w/v test item formulations were confirmed within 5%.

Duration of treatment / exposure:

(P) Males: 14 days before mating, 14 days/weeks during mating
(P) Females: 14 days/weeks before mating, 14 days/weeks during mating, 20 days/weeks during resulting pregnancies, 13 days through weaning of their F1 offspring.

Frequency of treatment:

Daily, 7 days per week.

Details on study schedule:

As per the OECD guideline.

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 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on repeated dose toxicity studies available to the Sponsor and given to the test facility.
- Rationale for animal assignment (if not random): Body weight-stratified randomisation.
- Fasting period before blood sampling for clinical biochemistry: not fasted

Positive control:

Not applicable.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day i.e. before and after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice per day i.e. before and after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Males on Day 1, 3, 7, 14, 21 , 28 of dosing; Females on Day 1, 3, 7, 14 of dosing, on Gestation Day 0, 7, 14, 20 and on post-natal Day 0, 4, 8, 13 and for non-copulated females on Day 21
All animals were weighed on necropsy day.

Oestrous cyclicity (parental animals):

Vaginal smear of all females were collected from Day 1 to Day 14 of dosing. The stages of oestrous cycle were determined with a light microscope after giemsa staining. The day from oestrous to the next oestrous were regarded as an oestrous cycle length and the mean oestrous length was calculated. When the oestrous was successive the first day was regarded as an oestrous.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, prostate weight, seminal vesicles weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; thyroid collected.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on next day after last dosing day
- Maternal animals: All surviving animals on next day after last dosing day or non-delivered females on gestation Day 24-26.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid

Statistics:

Data regarding body weights of parental animals, food consumption, T4 levels organ weights, body weights on necropsy, mean oestrous cycle length, pairing days until copulation, gestation length, number of implantation sites, number of pups born, number of live pups, body weights of pups, AGD, and number of nipples/areolae were analysed by the Bartlett's test for homogeneity of variance. If significant difference (p<0.05) was not noted, the values of the control group and each test substance group were analysed by the Dunnett's test. If significant difference (p<0.05) was noted in the Bartlett test, the values of the control group and test item group were analysed by the nonparametric Dunnett's test. Body weights of pups were calculated on each sex as sample unit for each litter.

Abnormal oestrous cyclicity, copulation index, conception index and delivery dam index were analysed by the Fisher's exact test between the control group and each test item group.

Indexes of delivery, birth, viability and sex ratio were examined by the Barlett's test. If significant difference (p<0.05) was not noted, the values of the control and test item groups were analysed by the Dunnett's test. If significant difference (p<0.05) was noted, the values of the control and test item group were analysed by the nonparametric Dunnett's test.

Reproductive indices:

- Copulation index (No. of copulated pairs/number of mated pairs) x 100
- Conception index (No. of pregnant females/No. of copulated pairs) x 100
- Delivery dam index (No. of pregnant females with live pups / no. of pregnant females) x 100
- Delivery Index (No. of pups born / No. of implantation sites) x 100

Offspring viability indices:

- Birth index (no. of live pups at birth/no. of implantation sites) x 100
- Viability index at birth (No. of live pups at birth/No. of pups at birth) x 100
- Sex ratio of pups at birth (No. of live males at birth/No. of live females at birth)
- Viability index on Day 4 after birth (No. of live pups on Day 4/ No. of live pups at birth) x 100
- Sex ratio of pups on Day 4 after birth (No. of live males on Day 4/No of live females on Day 4)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormal signs were observed in any males of any treatment group.

In females, treatment-related changes were not observed. Dam (No. 115) which whole pups died in the 62.5 mg/kg group showed staining on lower abdomen on postnatal Day 1. Dam (No. 127) which whole pups died in the 250 mg/kg group showed staining on lower abdomen on delivery day. These changes were not dose-related. NO abnormal changes were observed in the 1000 mg/kg group.

Dermal irritation (if dermal study):

not examined

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:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

T4 level was not affected in parental males and pups on postnatal Day 13 at any dose.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Spontaneous changes in males were as follows: unilateral spermatic granuloma in the epididymis in the control group, cyst formation in the pars intermedia of the pituitary in the 250 mg/kg group; unilateral spermatic granuloma in the epididymis in the 1000 mg/kg group. Macroscopic region in the Cowper's gland had no microscopic changes in the control and 65.2 mg/kg groups.

Spontaneous changes in females were erosion of the fundic mucosa of the glandular stomach in all test substance groups.

Non-delivered females in the control and 1000 mg/kg group had no microscopic lesions in the ovary. The 250 mg/kg group was not examined as there were no macroscopic lesions observed or treatment-related changes were observed in the high dose group.

Dams which whole pups died had the following changes: degeneration and necrosis of the tubular epithelium in the outer stripe of the outer medulla of the kidney in the 62.5 mg/kg group (No. 112); atrophy of the thymus in the 250 mg/kg group (No. 127). Animal no. 115 and 127 were not examined since no macroscopic lesions were observed or no treatment-related changes were observed in the high dose group.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Oestrous cycle stage of each dose group were as follows: metoestrus in 6/7, dioestrous in 1/7 of the control group; metoestrus in 4/8, dioestrous in 4/8 of the 62.5 mg/kg group; metoestrus in 5/6, dioestrous in 1/6 of the 250 mg/kg group; metoestrus in 6/8, dioestrous in 2/8 of the 1000 mg/kg group.

Non-delivered females (No. 103, 106 and 109) in control group showed metoestrus, prooestrous dioestrous, respectively. Non-delivered females (No. 124 and 130) in the 250 mg/kg group showed prooestrous. Non-delivered females (No. 134 and 135) in the 1000 mg/kg group showed prooetrous and metoestrous, respectively.

Dams (no. 112 and 115) which whole pups died in the 62.5 mg/kg group showed dioestrous. Dams (no. 125 and 127) which whole pups died in 250 mg/kg group showed dioestrous and prooestrous, respectively.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Delivery was not observed in 3 females of the control group, 2 females of the 250 mg/kg group, two females of the 1000 mg/kg group. No dose relationship in these changes.

Pairing days until copulation was increased in the 1000 mg/kg group. This change was limited to one pair of this group, no related changes in other parameters. Thus is was considered not toxicologically significant.

Oestrous cycle during the dosing period, copulation index, delivery index or delivery dam index were not affected in any test substance group.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No milk band, subnormal and emaciation were observed in the 62.5 mg/kg group. Subnormal temperature was observed in the 250 mg/kg group and one pup showed loss of right forelimb and loss of tail tip. No abnormal changes were observed in the 1000 mg/kg group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Deaths were observed in 1/96, 32/142 and 27/101 of the control, 62.5 and 250 mg/kg control groups, respectively.

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:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

T4 levels in pups was normal.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

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

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

See attachment for tables.

Applicant's summary and conclusion

Conclusions:

The NOAEL was 1000 mg/kg/day for reproduction and developmental toxicity under the condition of the study.