bis(2-ethylhexyl) 4,4’-{6-[4-tert-butylcarbamoyl)anilino]-1,3,5-triazine-2,4-diyldiimino}dibenzoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept-Dec 2022

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-450-8

EC Name:

-

Cas Number:

154702-15-5

Molecular formula:

C44H59N7O5

IUPAC Name:

2-ethylhexyl 4-[(4-{[4-(tert-butylcarbamoyl)phenyl]amino}-6-[(4-{[(2-ethylhexyl)oxy]carbonyl}phenyl)amino]-1,3,5-triazin-2-yl)amino]benzoate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

According to OECD test guideline 421, the preferred rodent species is the adult rat and SD rats are commonly used as laboratory rodents. Therefore, SD rats were used in this study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Feed: SPF rodent maintenance feed, Quantitative feeding
Animal Water: Reverse osmosis RO water, Continuous supply, not limited to free intake

Administration / exposure

Route of administration:

dermal

Vehicle:

water

Details on exposure:

The test item was prepared every day and used immediately after preparation. The test item was grounded in a mortar with specific amount of ultrapure water (1 g test item: 0.8 mL ultrapure water) to make the test item fully moist. The dosage were calculated according to the animals’ body weight once a week.

Preparation of Application Site: The dose area was at least 10% of the total body surface area

Test period:
Pre- exposure (PE): 14 days
Pre-mating (PM): 14 days
Mating (M): 1-3 days
Gestation (G): 22 days
Lactation (PND): 14 days
Dosing (male): 28 days

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 3 days
- Proof of pregnancy: Female rats were examined every morning for the presence of sperm or a vaginal plug until evidence of mating
- All females were mated successfully in three days.

Duration of treatment / exposure:

Exposure duration: The exposure lasted for 6 h a day.
Exposure period: The rats in treated group were dosed with the test item daily for 7 days a week from pre-mating period to lactation period. Male rats were dosed daily for a total 28-day dosing
period.
The control group was dosed with 0. 5 mL ultrapure water per rat per day.

Frequency of treatment:

Daily, 7 days a week, 6h a day

Doses / concentrations

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes

Examinations

Parental animals: Observations and examinations:

General clinical observations were performed once a day throughout the test period.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on the day of shaving and the day after shaving, and at the execution day. And females were weighed on the day of shaving, and PM0, PM7, M0, G0, G7, G14, PND0, PND4, PND11 and PND13

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption of female rats were measured once a week during pre- mating, pregnancy and lactation.
Food consumption of male rats were measured once a week during pre-mating.
- The average daily food consumption per animal in one week was calculated.

Oestrous cyclicity (parental animals):

PE period: Oestrous cycles were monitored to select females with regular cyclicity
PM and M period: Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating.
All dam rats were examined by vaginal smears in the morning on the day of necropsy to determine the stage of the oestrous cycles and allow with histopathology of ovaries

Sperm parameters (parental animals):

At least 200 sperm were counted consecutively and graded for each male rat, and analyzed by microscopy for each male rat.
The number of sperm head which were in the top left, top right, bottom left, bottom right and middle of the counting room were counted.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births.
Live pups were counted, sexed and litters weighed on PND0, PND4 and PND13 .
The AGD of each pup was measured on PND4. The number of nipples and areolas in male pups was counted on PND13.

Postmortem examinations (parental animals):

SACRIFICE
The adult rats anesthetized by intramuscular injection of Zoletil 50mg/mL at the dose of 50mg/kg body weight.
After blood was collected, the animal was sacrificed by means of bloodletting through the posterior abdominal
aorta.

GROSS NECROPSY
Male adult rats were euthanasia and gross necropsy on D28, unproduced female adult rats were euthanasia and gross necropsy on G25, and produced female adult rats were euthanasia and gross necropsy on PND13.

HISTOPATHOLOGY / ORGAN WEIGHTS
The ovaries, testes, accessory sex organs (uterus and cervix,
epididymides, prostate, seminal vesicles plus coagulating glands) , thyroid and all organs showing macroscopic lesions of all adult animals were preserved.
All weights were collected as soon as possible after dissection.

Detailed histological examination was performed on the ovaries of female adult, testicles and epididymides
of male adult animals in the treated group and control group.

Postmortem examinations (offspring):

GROSS NECROPSY
On PND13, all pups were carefully examined externally for gross abnormalities and internally for gross necropsy.

HISTOPATHOLOGY / ORGAN WEIGTHS
On PND13 , the thyroid from 1 male and 1 female pup per litter was preserved.

Statistics:

For measurement data ( included adult body weight, adult food consumption, pup body weight, pup body length, and pup AGD, T4), two independent samples T-test was used to determine which test item treated differed significantly from the solvent control. A significance level of 5% was used for all of the statistic analysis. T-test, assumed that the data were all with homogeneity of variance.
Comparison of two rates or two constituent ratios ( Normal Parturition Rate, sex ratio, Embryo Loss Rate, Malformation rate), the chi-square test was used.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

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:

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not specified

Details on results (P0)

For the pregnancy rate was 100%, all female rats in both treated group and control group were successfully conceived within 3 days.

There was no significant difference in parturition rate between the control group and the treated group and there was no significant difference in the sex ratio of fetal rats between the control group and the treated group.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Only one pups was dead on PND7 in control group, the body of the dead pup had bite mark, bite to dead by dam.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

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 specified

Details on results (F1)

For the thyroid hormones concentration ofpups at PND4 and PND 13, compared with the control group, there was no significant in the treated group.

For the birth of fetal rats, at PND0 , there were no significant difference between the control group and the treated group on rate of copulation success, normal parturition rate, abnormal rate, embryo loss rate), and no dead fetus in both groups.

There were no significant difference between the control group and the treated group on fetal survival rate at PND4. There were no significant difference between the control group and the treated group on pup mean weight at PND13, litter weight at PND13, the body length of pups PND13.
DHBT had no effect on the pups during the entire 13 - day development, and these indicators included: birth of surviving pups, dead pups and litter weight.

There was no abnormal fetal pathology case happened in any group, and there was no abnormal in the birth survival rate in the control group and treated group.

There was no significant difference in embryo loss rate between the control group and the treated group.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

viability

mortality

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

There was not any clear evidences of toxicity of Diethylhexyl Butamido Triazone at a dose level of 1000mg/kg body weight on male and female reproductive performance such as gonadal
function, mating behaviour, conception, development of the conceptus and parturition.

Executive summary:

No toxicity effect has been observed on reproductive/ growth and development for Diethylhexyl Butamido Triazone (DHBT) at the dose level of 1000 mg/kg bw.