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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to internationally recognised test methods. Some (minor) details on test method / results not available

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the 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): trioctyl benzene-1,2,4-tricarboxylate
- Molecular formula (if other than submission substance): C33 H54 O6
- Molecular weight (if other than submission substance): 546.79
- Smiles notation (if other than submission substance): c1(c(ccc(c1)C(OCCCCCCCC)=O)C(OCCCCCCCC)=O)C(OCCCCCCCC)=O
- InChl (if other than submission substance): 1S/C33H54O6/c1-4-7-10-13-16-19-24-37-31(34)28-22-23-29(32(35)38-25- 20-17-14-11-8-5-2)30(27-28)33(36)39-26-21-18-15-12-9-6-3/h22-23,27H,4-21,24- 26H2,1-3H3
- Structural formula attached as image file (if other than submission substance): see Fig. 89-04-3 Structure.png
- Analytical purity: > 99%
- Lot/batch No.: C-120
- Stability under test conditions: Stable

Test animals

Species:
rat
Strain:
other: Crj(SD) IGS, SPF CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Nippon Charles River Atsugi breeding centre
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 405 +/-16 g; Females: 256 +/- 18 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, CE-2. CLEA Japan.
- Water (e.g. ad libitum): yes, tap water via automatic watering system
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25
- Humidity (%): 50 - 65
- Air changes (per hr): 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 substance 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): 2mL/kg body weight
- Lot/batch no. (if required): V9F1299 supplied by Nacalai Tesque
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days (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 42 days
Females: from 14 days before pairing to day 4 of lactation
Frequency of treatment:
Daily during treatment periods - see above
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 30, 125, 500 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
13 males & 13 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of 14day preliminary study. Effects on body weight in males given 1000 mg/kg/day and increased liver weight in animals dosed at 500 and 1000 mg/kg
- 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 then weekly until sacrifice; Females: Predose then days 7, 14, 20; gestation days 1, 7, 14 & 20; lactation days 0 & 4.

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Males: Day 1-3, 7-8, 14-15, 29-30, 35-36 and 41-42. Females: Days 1-2, 7-8, 14-15; gestation days 7-8, 14-15 and 20-21; lactation days 3-4.

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 until confirmation of mating
Sperm parameters (parental animals):
Parameters examined in P males/group : yes - testis weight, epididymis weight
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
- Maternal animals: All surviving animals day 4 of lactation
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: epididymis, brain, heart, thymus, liver, kidney, spleen, adrenal glands, testes
Tissue preservation/microscopic examination: Brain, pituitary gland, spinal cord, thyroid, heart, trachea, bronchus, lung, liver, kidney, thymus, spleen, adrenal glands, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, bladder, mandibular lymph node, seminal vesicles (including coagulating gland), ovaries, vaginal lesions, uterus, prostate, mesenteric lymph nodes, sciatic nerve, femoral bone marrow, testes and epididymis preserved.
Histopathological examination performed on epididymis, testis and ovaries of all animals. Additionally examined was the liver of males of all groups and females of the high dose and control groups, the stomach of females of all groups and males of the high-dose and control groups. For the other preserved organs, those from 5 animals/sex/group were examined.


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:
Fisher's exact test - Changes in the frequency of animals’ sexual cycle, conception rate and copulation rate.
Mann-Whitney U test – Histopathological examination of the findings where the sum of the graded positive findings were significant difference between the control group by one-tailed test of exact probability of Fisher.
ANOVA by the Dunnet method of multiple comparisons or Kruskal-Wallis rank test in the case of non-uniform data – Other data
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:
effects observed, treatment-related
Description (incidence and severity):
One female at 500 mg/kg died Day 42. Salivation observed following dosing in males and females treated at 5oo mg/kg
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain in females treated at 500 mg/kg reduced day 7-14 of gestation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Body weight gain in females treated at 500 mg/kg reduced day 7-14 of gestation
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
reduced testis weight in animals treated at 125 mg/kg but with no dose-related trend
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
One female treated at 500 mg/kg died on Day 42 (Day 23 of gestation). Salivation immediately after dosing was seen intermittently from the 14th day and periocular brown stating was observed prior to day of death.
In surviving animals, salivation immediately after dosing was observed in 4 males and 11 females treated at 500 mg/kg. This was seen intermittently from the 9th day of dosing, was transitory and disappeared 2-3 hours following dosing. Alopecia, generalised staining around the vulva, emaciation and loose stools were observed in individual animals.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
In males no significant difference between the control group and the test substance treated groups was apparent.
In females, significant differences were not observed in body weight although body weight change in the period Day 7-14 of gestation was significantly lower compared to controls in animals dosed at 500 mg/kg.

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

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No effects observed on oestrus cycle, mating indices.
One female treated at 500 mg/kg died during the gestation period. Other than this there was no significant difference between the control group and the test substance administration group(s). The number of corpora lutea and the number of implantation sites was not significantly different between the control group and the test substance administered group(s). Parturition and lactation state were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS):
In males, a significant reduction in testis weight was observed in the group treated at 125 mg/kg, but this trend was not dose-related.
A significant increase in liver weight was observed in females treated at 125 and 500 mg/kg, relative liver weight also showing a statistically significant difference in the 500 mg/kg group.
The relative weight of the heart was significantly lower than the control group in females of all treatment groups compared with controls. Absolute weight of this organ change showed no significant variation.

GROSS PATHOLOGY (PARENTAL ANIMALS):
As abnormalities of the reproductive organs, 1 male treated at 125 mg/kg and 1 males treated at 500 mg/kg unilateral miniaturisation of the epididymis was observed.

HISTOPATHOLOGY (PARENTAL ANIMALS):
As abnormalities of the reproductive organs, 1 male treated at 125 mg/kg and 1 male treated at 500 mg/kg showed unilateral atrophy of the seminiferous tubules while bilateral atrophy was noted in 1 male treated at 30 mg/kg and 1 male of the control group. Reduced sperm numbers were noted in the lumen of the epididymis of 1 male of the control group, 1 male treated at 125 mg/kg and 1 male treated at 500 mg/kg.
Cell debris was noted in the lumen of the epididymis with the same incidence. A spermatic granuloma was observed in the left side of the epididymis of 1 male treated at 500 mg/kg. Interstitial lymphocytic infiltration was noted in the epididymis of 2 control males, 2 males treated at 30 mg/kg, 1 male treated at 125 mg/kg and 1 male treated at 500 mg/kg. Interstitial/epithelial lymphocytic and plasma cell infiltration was noted in the ventral prostate of 4 control males and 2 males treated at 500 mg/kg (intermediate groups not examined).
Ovaries of female animals were not remarkable.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: For effects on reproductive and developmental toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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

Details on results (F1)

VIABILITY (OFFSPRING):
Neonatal sex ratio on Days 0 and 4 of lactation was significantly different between controls and animals treated at 500 mg/kg. No significant differences were apparent in the number of infants, calving rate, the rate of live births.

CLINICAL SIGNS (OFFSPRING):
No effects

BODY WEIGHT (OFFSPRING):
Body weight of male and female pups on Days 0 and 4 of lactation were not remarkable.

GROSS PATHOLOGY (OFFSPRING):
There was no significant difference between the control and treated groups on external observation of pups and no abnormalities were observed at autopsy of pups.

HISTOPATHOLOGY (OFFSPRING):
Not examined


Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

See attached document (89 -04 -3 tabulated data.pdf)

Applicant's summary and conclusion

Conclusions:
Treatment with the substance had no significant effects on reproductive & developmental toxicity. The NOELs are considered to be 500 mg/kg/day for both reproductive toxicity and developmental toxicity.

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 a repeat-dose toxicity combined with a screening study of reproductive toxicity on read-across substance, 1,2,4 -benzene

tricarboxylic acid, trioctyl ester, no significant effect on reproductive ability, organ weight or histopathology of the ovary, delivery or maternal behaviour was apparent. The NOEL for reproductive / developmental toxicity is considered to be 500 mg/kg/day for both parents and offspring, the highest dose level examined.