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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:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
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

Constituent 1
Chemical structure
Reference substance name:
Trioctyl benzene-1,2,4-tricarboxylate
EC Number:
201-877-4
EC Name:
Trioctyl benzene-1,2,4-tricarboxylate
Cas Number:
89-04-3
Molecular formula:
C33H54O6
IUPAC Name:
trioctyl benzene-1,2,4-tricarboxylate
Details on test material:
- Name of test material (as cited in study report): 1,2,4-benzenetricarboxylic acid, trioctyl ester (BCTO)
- 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): CCCCCCCCOC(=O)c1ccc(c(c1)C(=O)OCCCCCCCC)C(=O)OCCCCCCCC
- 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
- Substance type: pale yellow clear liquid
- Physical state: liquid
- Analytical purity: >= 99.0%
- Lot/batch No.: no data
- Stability under test conditions: Stated to be stable
- Storage condition of test material: Ambient conditions

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan's Atsugi Breeding Center
- Age at study initiation: 10 weeks;
- Weight at study initiation: Males: 377.1-433 g; Females: 225.2-238.8 g
- Fasting period before study: No
- Housing: wire mesh cages, in pairs for mating; From 18 days gestation onwards (day of confirmed copulation = gestation day 0), females were housed in plastic rat breeding cages (w: 350w × d: 400 × h: 180 mm) with paper pulp chip bedding (Alpha-dri, Kasho Co., Ltd.).
- Diet: pelleted diet (CE-2 from Clea Japan Inc.), ad libitum
- Water: tap water, ad libitum
- 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

IN-LIFE DATES: From: May 2, 2000 To: June, 2000

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: weekly, dissolving of required weight of the substance in corn oil, thereafter kept in a refrigerator in airtight containers in the dark until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance is poorly soluble in water
- Amount of vehicle (if gavage): 2ml/kg bw
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
- Any other deviations from standard protocol: None reported
- After successful mating each pregnant female was caged (how): single per cage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration of preparations were analysed and the results were in the range 98-102% of nominal. Formulated substance stable for at least 8 days.
Duration of treatment / exposure:
males: 42 days
females: 2 weeks prior mating, through mating, gestation and up to lactation days 4 (min 40 days to 53 days)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
low dose
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
mid dose
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
high dose
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of preliminary study (14 days repeated oral toxicity study using concentrations of 0, 500 and 1000 mg/kg bw/d - effects on body weight, increased liver weight and oedema of gastric mucosa observed in the high dose group)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were included: daily,during administration period observations were made twice or more per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for signs of ill health/moribund condition and response to treatment

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Weekly; Females: days 0 (day of administration) , 7, 14, gestation days 1, 7, 14, 20 and parturition, lactation days 0 and 4 and days of necropsy.

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 (supplied and remaining food were measured on administration days)
- Males: days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42;
- Females: days 1-2, 7-8, 14-15; days 0-1, 7-8, 14-15 and 20-21 of gestation, days 3-4 of lactation
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No - Not applicable as animals dosed by gavage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not applicable (no dietary study)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable (no drinking water study)
Oestrous cyclicity (parental animals):
examination pre-dose and during dosing period
Sperm parameters (parental animals):
- testis weight and microscopic pathology, epididymis weight and microscopic pathology
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 following 42 days of treatment
- Maternal animals: All surviving animals day 4 of lactation

GROSS NECROPSY
Yes (Macroscopic examination of organs and tissues including organ weights: brain, heart, thymus, liver, kidneys, spleen, adrenal gland, and for males testis and epididymis. In females particular attention paid to uterus and ovaries (number of implantations number of corpus lutea and, under a stereoscopic microscope, implantation scars.)

HISTOPATHOLOGY / ORGAN WEIGHTS
Yes (Testes and epididymides (all males), liver (all males and females of control, mid and high dose groups) stomach (all females and males of high dose and control groups). For other organs 5 males/5 females examined from high dose and control groups)
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:
The frequency of animals with changes to the estrous period, the copulation index and fertility rate were analysed by Fisher’s exact test.
Clinical signs and histopathological findings were graded by Mann-Whitney U test with the sum of the positive findings. Significant difference tests were performed between the control group by one-sided test of direct probability of Fischer.
For other data obtained for each specimen, or each 1 sample in the case of mean litter value, uniformity of distribution for each group was first obtained by Bartlett method. In case of uniform distribution, one-way analysis of variance was conducted and, where there was significance between groups, multiple comparison testing was conducted by Dunnett method. However, where distribution was 0 for any of the groups and where distribution was not uniform, Kruskal Wallis rank test was performed, and where significance was found between groups, multiple comparison testing was conducted by Dunnett method. Significance level was set at 5% in all cases.
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 generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
1 female in the 500 mg/kg administered group died following administration on administration day 42 (gestation day 23). In addition to salivation after dosing, which was seen intermittently from day 14, to administration, on the day of death, red discharge and soiled fur around the eyelid were observed.
In surviving animals salivation was observed immediately after administration in 11 males and 4 females in the 500 mg/kg group. Salivation was seen intermittently early on in the animals from day 9 of administration, but in all of these animals it disappeared 2-3 hours after administration and was a temporary change. Other than this, 2-5 males and females showed fur loss in each administration group, and 1 female in the control group was observed with emaciation, loose stool and soiled perianal region
Mortality:
mortality observed, treatment-related
Description (incidence):
1 female in the 500 mg/kg administered group died following administration on administration day 42 (gestation day 23).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
For males, no significant difference was found for actual body weight and weight increase between control and treatment groups. For females, no significant difference was found for actual body weight, but comparison of the control group with the 500 mg/kg group showed a significant reduction in body weight increase for gestation days 7-14.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Concerning food consumption, there were no significant difference between control and treatment groups.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
changes in urinalysis, haematology and clinical chemistry parameters are discussed in detail in section 7.5.1 and are not thought to influence any of the investigated parameters in this reproductive/developmental toxicity screening study
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
changes in urinalysis, haematology and clinical chemistry parameters are discussed in detail in section 7.5.1 and are not thought to influence any of the investigated parameters in this reproductive/developmental toxicity screening study
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
changes in urinalysis, haematology and clinical chemistry parameters are discussed in detail in section 7.5.1 and are not thought to influence any of the investigated parameters in this reproductive/developmental toxicity screening study
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Hepatocellular hypertrophy centrilobular region of liver in males treated at 500 mg/kg/day
(Mild hypertrophy of hepatocytes the centrilobular region of the liver was observed in five males treated at 500 mg/kg/day. Other effects observed in indiviual animals and which are not judged as adverse are discussed in detail in section 7.5.1 and are not thought influence any of the investigated parameters in this reproductive/developmental toxicity screening study.)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on reproductive performance or sex organs

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

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on pup weight, sex ratio, survival index, viability index and no abnormalities found in visual examination of live pups or necropsy of pups at the end of the study

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Oestrus cycle

 

Dose level (mg/kg body weight/day)

0

30

125

500

4 day cycle

12

13

13

13

Irregular cycle

1

0

0

0

Length of oestrus cycle (days

4.1 ± 0.2 (13)

4.0± 0.0 (13)

4.0 ± 0.0 (13)

4.0 ± 0.0 (13)

   

Reproductive performance

 

Dose level (mg/kg body weight/day)

0

30

125

500

Number of mated pairs

13

13

13

13

Number of copulated pairs

13

13

13

13

Copulation index

100

100

100

100

Number of pregnant animals

12

13

13

13

Fertility index

92.3

100

100

100

Number of pairing days

2.3 ± 1.3 (13)

2.5 ± 1.1 (13)

1.8 ± 1.0 (13)

2.2 ± 1.1 (13)

Frequency of oestrus (mean times during mating)

1.0 ± 0.0 (13)

1.0 ± 0.0 (13)

1.0 ± 0.0 (13)

1.0 ± 0.0 (13)

Developmental toxicity parameters

 

Dose level (mg/kg body weight/day)

0

30

125

500

Number of pregnant animals

12

13

13

13

Number of pregnant animals with live pupsg

12

13

13

12 *

Gestation index

100

100

100

92.3

Gestation length (days)

22.6 ± 0.5

22.2 ± 0.4

22.5 ± 0.5

22.6 ± 0.5

Number of corpora lutea

17.3 ± 1.5

17.9 ± 2.5

17.0 ± 1.8

16.9 ± 2.3

Number of implantation sites

15.8 ± 1.5

16.3 ± 1.4

15.8 ± 1.3

15.2 ± 2.6

Implantation index

91.6 ± 7.8

91.6 ± 6.5

93.6 ± 6.1

90.3 ± 12.8

 

 

 

 

 

Lactation day 0:

 

 

 

 

Number of pups born

14.7 ± 2.3

15.2 ± 1.9

14.7 ± 1.4

13.3 ± 4.0

Delivery index

92.8 ± 7.4

93.3 ± 7.1

92.9 ± 6.5

88.1 ± 20.7

Number of pups alive

14.1 ± 1.8

15.1 ± 1.8

14.5 ± 1.4

13.2 ± 4.0

Birth index

89.5 ± 7.8

92.4 ± 7.3

91.9 ± 6.8

86.9 ± 20.1

Live birth index

96.6 ± 6.3

99.1 ± 2.3

99.0 ± 2.5

98.8 ± 2.8

Pup weight (g)   - Males

7.1 ± 0.8

6.8 ± 0.5

7.1 ± 0.8

7.2 ± 1.0

                          - Females

6.7 ± 0.8

6.4 ± 0.5

6.5 ± 0.7

6.9 ± 1.0

Sex ratio (% males)

45.3 ± 12.9

53.5 ± 12.3

46.6 ± 11.5

53.0 ± 18.2

 

 

 

 

 

Lactation day 4:

 

 

 

 

Number of live pups

13.8 ± 1.7

14.8 ± 1.6

14.1 ± 1.1

12.9 ± 3.9

Viability index

98.3 ± 4.1

98.6 ± 2.7

98.6 ± 3.6

98.4 ± 5.4

Pup weight (g)   - Males

10.6 ± 2.6

10.5 ± 1.5

11.1 ± 1.6

11.5 ± 2.3

                          - Females

10.2 ± 2.7

10.0 ± 1.3

10.4 ± 1.4

11.1 ± 2.3

Sex ratio (% males)

44.8 ± 12.4

53.2 ± 12.4

46.3 ± 11.4

53.5 ± 17.4

* One female found dead on gestation day 2

Applicant's summary and conclusion

Conclusions:
Combined repeated dose toxicity study with reproduction/developmental toxicity screening was conducted for 1,2, 4 benzenetricarboxlyic acid, trioctyl ester (C8TM). This GLP conform study was performed according to OECD testing guideline 422. Sprague-Dawley rats (13 animals each/group) were treated with 0, 30, 125 and 500 mg/kg for 42 days for males and for pregnant females throughout gestation until day 4 of lactation. No adverse effects on oestrous cycle, copulation, fertility, delivery or lactation and no changes related to gestation index, gestation length, numbers of corpora lutea, implantation sites or implantation index. There were no changes in sex ratio, body weight, viability or morphology of pups. The No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 500 mg/kg/day for both parent animals and offspring, this being the highest dose level investigated.
Executive summary:

Combined repeated dose toxicity study with reproduction/developmental toxicity screening was conducted for 1,2, 4 benzenetricarboxlyic acid, trioctyl ester at 0 (carrier), 30, 125 and 500 mg/kg for both female and male animals, with repeated administration in Sprague Dawley female and male rats (13 animals each/group) from 2 weeks before mating up until mating, then for 42 days for males and for pregnant females throughout gestation until day 4 of lactation. This GLP conform study was performed according to OECD testing guideline 422.

Effects on parental animals concerning repeated dose toxicity are presented in detail in the respective endpoint study record and will not be discussed further. The NOAEL for systemic effects after repeated substance administration is judged to be 125 mg/kg bw/day, based on changes in clinical chemistry in both sexes.

For reproduction and developmental toxicity screening, no changes suggestive of effect from the test substance were noted in female estrous cycle, male and female copulation index, or fertility index, delivery and lactation conditions in dams. Furthermore, no effect from the test substance was noted in the birth index, gestation period, numbers of corpora lutea, number of implantation sites and implantation index. In addition, no changes caused by the test substance were noted for viability, sex ratio and body weight of pups. Thus the NOAEL reproduction/fertility/developmental for parental animals and offspring is judged to be 500 mg/kg bw/day (highest dose tested).

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