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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Tris (2-ethylhexyl) 1,2 4-benzenetricarboxylic acid
IUPAC Name:
Tris (2-ethylhexyl) 1,2 4-benzenetricarboxylic acid
Details on test material:
- Name of test material (as cited in study report): Tris (2-ethylhexyl) 1,2, 4-benzenetricarboxylic acid
- Physical state: liquid
- Analytical purity: 99.0%
- Lot/batch No.: N-80301
- Expiration date of the lot/batch:
- Stability under test conditions: stable, confirmed by manufacturer
- Storage condition of test material: room temperature in airtight opaque containers in the dark

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: (P) x 10 weeks for both sexes;
- Weight at study initiation: (P) 373-435 g for males; 217-257 g for females
- Fasting period before study: No
- Housing: wire mesh cages, in pairs for mating
- Use of restrainers for preventing ingestion (if dermal): Not applicable
- Diet (e.g. ad libitum): yes, pelleted diet, CRF-1. Oriental Yeast Co. Ltd.
- Water (e.g. ad libitum): yes, tap water via automatic watering system
- Acclimation period: 14 days for males; 10 days for females

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/- 3
- Humidity (%): 55+/-10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES:
-Terminal kill: Male day 47, Female day 4 of lactation

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared weekly by dissolving required weight of tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate in corn oil and stored in airtight containers in the dark until use.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate is poorly soluble in water
- Concentration in vehicle: As required to achieve nominal dose
- Amount of vehicle (if gavage): 5 ml/kg

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: within the limit of 14 days until proof of pregnancy (until sperm detected in vagina).
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged singly
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Not reported
Duration of treatment / exposure:
Males: 46 days;
Females: from 14 days before mating to day 3 of lactation
Frequency of treatment:
Once a day during treatment period
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle
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:
12 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of 14 day preliminary study. Effects on body weight in animals given 1000 mg/kg/day.
- Rationale for animal assignment: Random, stratified body weight
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:General appearance once a day for parents and foetuses after birth

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Predose, 2, 5, 7, 10 and 14 days and weekly thereafter until sacrifice; Females: Predose, 2, 5, 7, 10 and 14 days. Gestation period: 0, 1, 3, 5, 7, 10, 17 and 20 days. Lactation period: 0,1 and 4 days. During cohabitation period, the same day with male

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - at same intervals as body weight except lactation period and day of sacrifice for males and 0 day of gestation and lactation for females.
Oestrous cyclicity (parental animals):
Examined pre-dose and during dosing period
Sperm parameters (parental animals):
Parameters examined in 5 P males/group : yes
[testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm morphology
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 and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

WEIGHTS:
Day 0 and 4
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals 47 days
- Maternal animals: All surviving animals day 4 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations of all organs and including the cervical, thoracic, and abdominal viscera. Parents: all organs; Pups: all organs
Count: Implantation sites and corpus luteum of ovary of all animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following tissues were prepared for microscopic examination and weighed, respectively: organ weights: males testis and epididymis; females ovary
- Microscopic examination: Testis and epididymis, count of sertoli cells, spermatocytes, round and elongated spermatids in seminiferous tubules of 5 animals /group (Stage I-VI, VII-VIII, IX-XI, XII-XIV of spermatozoon formative cycle).
Females: Ovary
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination).
Pups: found dead and abnormalities

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Gross pathlogy of all organs were tested. Dead pups and abnormal organs were tested histopathogy.
Statistics:
Chi squared test for 1 grade positive data and Fisher's test for another. Bartlett's test or Kruskal Wallis' test for 2 or more positive grade data. Dunnett's test or Mann-Whitney U test used for examination.
Reproductive indices:
Copulation Index: No. of pairs with successful copulation/no. of pairs mated X 100
Fertility Index: No of pregnant females/no. of pairs with successful copulation X 100
Implantation index: No. of implantation sites/no. of corporea lutea X 100
Delivery index: No. of pups born/no. of implantation sites X 100
Gestation index: No. of females with live pups delivered / no. of pregnant females X 100
Nursing index: No. of females nursing live pups / no. of females with normal delivery X 100
Offspring viability indices:
Live birth index: No. of live pups at birth / No. of pups at birth X 100
Viability index: No. of live pups on day 4 / No.of live pups at birth

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistical significant difference from controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistical significant difference from controls.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry 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, treatment-related
Description (incidence and severity):
Slightly reduced numbers of spermatocytes and spermatids in 2/12 and 11/12 animals given 300 and 1000 mg/kg/day tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate respectively and a moderate decrease in 1/12 animals given 1000 mg/kg/day tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate. At this animal, a few multinucleate giant cell were appeared and slightly vacuolization of sertoli sells were observed. Also, at the epididymis, moderate amount of cell debris moderate decrease of spermatids and slightly granuloma of spermatic were observed. In addition the number of cells/number of spermatids in seminiferous tubules was reduced in males given 300 mg/kg/day tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate in stages I-VI. In males given 1000 mg/kg/day in stage I-IV numbers of spermatocytes and spermatids were reduced. In stages VII-XIV spermatocyte and spermatid numbers continued to be low and the sertolicell ratio was also reduced.

For the control group, atrophy of seminiferous tubule were observed 2 animals. At these animals, slightly amount of cell debris were observed. one of these animals, slight decrease of spermatids was also observed.

For females:
Cyst of corpus luteum of ovary was observed 2 animals of 300 mg/kg dosing group. No abnormal ovary observed at the female of 100 mg/kg dosing without successful copulation, females of control and 100 mg/kg dosing without pregnant.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No statistical significant difference from controls.
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain in pups of the 300 mg/kg/day group for both sexes were slightly low. However as the body weight and body weight gain in the 100 and 1000 mg/kg/day groups were unaffected this was not considered to be a definite effect of treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
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)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Repeat dose toxicity: Histopathological examination revealed reduced spermatocytes and spermatids in the testes of males given tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate at doses of 300 and 1000 mg/kg/day. Treatment with tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate had no effect on the appearance, condition or behaviour, body weight, food consumption, necropsy findings, weights of the testes, epididymis or ovaries, or histopathology of the ovaries. The NOELs are considered to be 100 and 1000 mg/kg/day for males and females respectively.

Reproductive and developmental toxicity: With the exception of the effects in male described above treatment with tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate at dosages of 100, 300 or 1000 mg/kg/day had no effect on reproductive ability, organ weight or histopathology of the ovary, delivery or maternal behaviour of the dams. The NOELs are considered to be 100 and 1000 mg/kg/day for males and females respectively.

Pup post natal development: No effects of treatment with tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate at dosages of 100, 300 or 1000 mg/kg/day were detected on viability, general appearance, body weights or autopsy findings. The NOEL is considered to be 1000 mg/kg/day for males and female offspring.
Executive summary:

An OECD reproductive/developmental toxicity screening test [TG 421] was performed (MHW Japan, 1998) in compliance with GLP criteria and this study was identified to have been well conducted and reported.

A gavage study in SD rats was conducted at doses of 100, 300 and 1,000 mg/kg/day (male: 46 days, female: from 14 days before mating to day 3 of lactation) of tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate.

Histopathological examination revealed decreased spermatocytes and spermatids in the testes of males given the substance at doses of 300 or 1000 mg/kg/day. Treatment had no effect on the appearance, body weight, food consumption, necropsy findings, weights of the testes, epididymis or ovaries, or histopathology of the ovaries. On the basis of these findings, the NOELs for systemic toxicity are considered to be 100 and 1000 mg/kg/day for males and females respectively.

With the exception of the effects in males observed on histopathological examination, treatment at dosages of 100, 300 and 1000 mg/kg/day had no effect on reproductive ability, organ weight or histopathological appearance of the ovaries, delivery or maternal behaviour of the dams. No effect of tris(2-ethylhexyl) benzene-1,2,4-tricarboxylatewas detected on viability, general appearance, body weight or autopsy findings of offspring. Body weight gain of pups at 300 mg/kg bw/day was slightly low, but body weights of all pups at 100 and 1000 mg/kg bw/day were not statistically different form control. On the basis of these findings, the NOELs for reproductive / developmental toxicity were considered to be 100mg/kg bw/day for male rats, 1,000 mg/kg bw/day for female rats, and 1,000 mg/kg bw/day for offspring.