Trioctyl benzene-1,2,4-tricarboxylate

Toxicity to reproduction

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

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

Test material information

Constituent 1

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:

1,2,4-trioctyl benzene-1,2,4-tricarboxylate

Test material form:

liquid

Specific details on test material used for the study:

- Lot/batch No.: C-120

Test animals

Species:

rat

Strain:

other: 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;
- Weight at study initiation: (P) Males: 388-420 g; Females: 242-277 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
- Water (e.g. ad libitum): yes, tap water
- 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 the substance in corn oil and stored refrigerated in airtight containers in the dark until use.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Substance is poorly soluble in water
- Concentration in vehicle: As required to achieve nominal dose, up to 25% w/v
- Amount of vehicle (if gavage): 2mL/kg

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 except that 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: from 14 days before pairing for 42days
Females: from 14 days before pairing to day 4 of lactation

Frequency of treatment:

Daily during treatment period

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 30, 125 and 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 14 day preliminary study at dose levels of 500 and 1000 mg/kg/day. Effects on body weight, increased liver weight and oedema of gastric mucosa noted 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


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

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 - Males: days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42; Females: days 1-2, 7-8, 14-15, days 7-8, 14-15 and 20-21 of gestation, 3-4 of lactation

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Oestrous cyclicity (parental animals):

Examined pre-dose and during dosing period

Sperm parameters (parental animals):

Parameters examined in P males/group : yes
- 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
- 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 weighed: Brain, heart, thymus, liver, kidneys, spleen, adrenals, testes, epididymides
The following tissues were prepared for microscopic examination: Brain, heart, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, stomach, prostate, urinary bladder, lungs (and bronchi), ileum, trachea, mandibular lymph node, seminal vesicles and coagulating gland, duodenum, jejunum, caecum, colon, rectum, mesenteric lymph nodes, pituitary gland, thyroid gland, sciatic nerve, spinal cord, bone marrow (from femur), ovary, uterus, vagina

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:

Sexual cycle, copulation rate and conception rate were examined using 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.
Other data, as a sample value or the average value of each litter, were obtained for each individual and tested for uniformity of the variance of each group by the method of Bartlett. If the distribution was uniform, ANOVA and significance was examined using the multiple comparison method of Dunnett. If the distribution was not uniform, a Kruskal-Wallis rank test was performed with significance between the groups examined by Dunnett test. The significance level was 5%.

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

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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 of liver of males at 500 mg/kg/day

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No deaths occurred except for one female treated at 500 mg/kg/day which died on day 23 of gestation. Temporally increase in salivation after dosing was observed in animals treated at 500 mg/kg/day.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Decreased body weight gain from day 7 to 14 of gestation observed in females treated at 500 mg/kg/day. No adverse effects on body weights in males. No effects on food consumption in either sex.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): No data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No effects

ORGAN WEIGHTS (PARENTAL ANIMALS): No effects

GROSS PATHOLOGY (PARENTAL ANIMALS): No effects

HISTOPATHOLOGY (PARENTAL ANIMALS): Hypertrophy of hepatocytes in the centrilobular zone of the liver in males treated at 500 mg/kg/day. No adverse changes in other organs.

Effect levels (P0)

Dose descriptor:

LOEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

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): No effects

CLINICAL SIGNS (OFFSPRING): No effects

BODY WEIGHT (OFFSPRING): No effects

GROSS PATHOLOGY (OFFSPRING): No effects

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: Absence of effects on pup weight; sex ratio; survival index; viability index

Target system / organ toxicity (F1)

Critical effects observed:

no

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	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 young	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 23

Applicant's summary and conclusion

Conclusions:

A repeated-dose toxicity combined with a reproductive/developmental toxicity screen conducted according to OECD Test Guideline No 422 found 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 (NOEL) 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:

A repeated-dose toxicity combined with a reproductive/developmental toxicity screen conducted according to OECD Test Guideline No 422 found 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 (NOEL) 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.