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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to internationally recognised test methods. For read across justification see Section 13.

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
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2, 4-benzenetricarboxylic acid trioctyl ester
- 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): O=C(OCCCCCCCC)c(ccc(c1C(=O)OCCCCCCCC)C(=O)OCCCCCCCC)c1
- InChl (if other than submission substance): 1S/C27H42O6/c1-3-5-7-9-11-12-14-16-20-33-27(31)23-18-17-22(21-24 (23)25(28)29)26(30)32-19-15-13-10-8-6-4-2/h17-18,21H,3-16,19-20H2,1-2H3, (H,28,29)
- Structural formula attached as image file (if other than submission substance): see 89-04-3 structure.png

- Analytical purity: =>99%
- Lot/batch No.: C-120
- Storage condition of test material: Ambient conditions

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 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 studyat 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 animals)

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
Description (incidence and severity):
Increased liver weight in females at 125 and 500 mg/kg/day
Gross pathological findings:
no effects observed
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:
NOEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of 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

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

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.