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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tris (2-ethylhexyl) 1,2, 4-benzenetricarboxylic acid
- Structural formula attached as image file (if other than submission substance): see Fig.
- 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:
other: Crj:CD:SD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: (P) x 10 wks;
- Weight at study initiation: (P) Males: 373-435 g; Females: 217257 g
- 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: 14d for males; 10d for females

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared weekly by dissolving required weight of TOTM 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): TOTM is poorly soluble in water
- Concentration in vehicle: As required to achieve nominal dose
- Amount of vehicle (if gavage): 5ml/kg
- Lot/batch no. (if required): No batch number reported but supplied by Katayama Chemical
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 d (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 (how): singly
- Any other deviations from standard protocol: None reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
Males: from 14 days before pairing for 46d;
Females: from 14d before pairing to d3 of lactation
Frequency of treatment:
Daily during treatment periods see above
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control) 100 300 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
12 males & 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of 14d preliminary study. Effects on body weight in animals given 1000 mg/kg/day
- Rationale for animal assignment (if not random): Random, stratified body weight
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, parents & foetuses

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Predose, 2, 5, 7, 10 & 14d & weekly thereafter until sacrifice; females: Predose, 2, 5, 7, 10 & 14d. Gestation period: 0, 1, 3, 5, 7, 10, 17 & 20d. Lactation period: 0,1 & 4d.

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 0d of gestation & lactation for females.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:
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
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals 1d after mating
- Maternal animals: All surviving animals d4 of lactation; if did not mate 1d after mating period ended
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations of all organs & including the cervical, thoracic, and abdominal viscera. Pups: all organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively. : organ weights: males Testis & epididymis; females ovary;
microscopic examination: Testis & epididymis, sertoli cell count, spermatocytes, round & elongated spermatids in seminiferous tubules of 5 animals /group (Stage 1-VI, VII-Viii, IX-XI, XII-XIV of spermatoan 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 & abnormalities

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Chi squared test for 1 grade positive data and Fisher's test for other. Bartlett's /Kruskal Wallis' test for 2 or more positive grade data. Dunnett's test or Mann-Whitney U test for assessment.
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 d4/no.of live pups at birth

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
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
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below for males
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
See below for males
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No effects

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): No effects

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): See histopathology (below)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No effects

ORGAN WEIGHTS (PARENTAL ANIMALS): No effects

GROSS PATHOLOGY (PARENTAL ANIMALS): No effects

HISTOPATHOLOGY (PARENTAL ANIMALS): Slightly reduced numbers of spermatocytes & spermatids in 2/12 & 11/12 animals given 300 & 1000 mg/kg/day TOTM respectively and a moderate decrease in 1/12 animals given 1000 mg/kg/day TOTM. In addition the number of cells/number of spermatids in seminiferous tubules was reduced in males given 300 mg/kg/day TOTM in stages I-VI. In males given 1000 mg/kg/day in stage I-IV numbers of spermatocytes &spermatids were reduced. In stages VII-XIV spermatocyte & spermatid numbers continued to be low & the sertolicell ratio was also reduced.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Histopathology; sperm characterization, numbers & ratio of sertoli cells in males given 1000 mg/kg/day TOTM & reduced numbers of spermatids in animals given 300 mg/kg/day.
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'
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of effects on pup weight; sex ratio; survival index; viability index following treatment of parents with TOTM at dosages of 100, 300 or 1000 mg/kg/day.
Remarks on result:
other: Generation: pups (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
slightly low bodyweights & body weight gain in pups in 300 mg/day/kg group. No difference, relative to controls, at 100 and 1000 mg/kg b.w.
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
Description (incidence and severity):
Pups found dead or with abnormalities only

Details on results (F1)

VIABILITY (OFFSPRING): No effects

CLINICAL SIGNS (OFFSPRING): No effects

BODY WEIGHT (OFFSPRING):Body weight & body weight gain in pups from the 300 mg/kg/day group were slightly low. However as the body weight & body weight gain in the 100 & 1000 mg/kg/day groups were unaffected this was not considered to be a definite effect of treatment.

GROSS PATHOLOGY (OFFSPRING): No effects

HISTOPATHOLOGY (OFFSPRING): No effects

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

See attached document (MITI Repro sreen.pdf)

Applicant's summary and conclusion

Conclusions:
Repeat dose toxicity: Histopathological examination revealed reduced spermatocytes & spermatids in the testes of males given TOTM at doses of 300 or 1000 mg/kg/day. Treatment with TOTM 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 & 1000 mg/kg/day for males & females respectively.
Reproductive & developmental toxicity: With the exception of the effects in male described above treatment with TOTM 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 & 1000 mg/kg/day for males & females respectively.

Pup post natal development: No effects of treatment with TOTM 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 & female offspring.
Executive summary:

In an OECD screening study of reproductive toxicity histopathological examination revealed reduced spermatocytes & 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, condition or behaviour, body weight, food consumption, necropsy findings, weights of the testes, epididymis or ovaries, or histopathology of the ovaries. The NOELs for systemic toxicity are considered to be 100 & 1000 mg/kg/day for males & females respectively.

With the exception of the effects in males, treatment 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 NOEL for reproductive / developmental toxicity is considered to be 1000 mg/kg/day for offspring.