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screening for reproductive / developmental toxicity
Data waiving:
other justification
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Reason / purpose for cross-reference:
data waiving: supporting information
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
November to December 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Analogue Approach
Reason / purpose for cross-reference:
read-across source
according to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
Limit test:
Details on test animals or test system and environmental conditions:
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: female approx. 11 weeks (male 13 weeks)
- Weight at study initiation: 221-260 g female, males at least 307 g
- Fasting period before study: no
- Housing: no more than 5 of one sex to a cage, in clear polycarbonate cages (43 x 27 x 18 cm) with a stainless steel mesh lid and floor
- Diet (e.g. ad libitum): commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese, Italy) ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: approximately 2 weeks before the start of the treatment

- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15 to 25
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2009-11-12 To: 2009-12-14
Route of administration:
oral: gavage
corn oil
Details on exposure:
The required amount of 1,2,4-Benzenetricarboxylic acid, decyl octyl ester will be
dissolved/suspended in the vehicle. The formulation will be prepared daily. Concentrations will be calculated and
expressed in terms of test item as supplied.

- Justification for use and choice of vehicle (if other than water): no justification given
- Concentration in vehicle: 20, 60, 200 mg/ml (formulation analysis: 19-21, 60, 216-217 mg/ml)
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The proposed formulation procedure and the stability at 24 hours at room temperature were verified in RTC Study no. 77280 in the range of 10 - 200 mg/mL to confirm that the method was acceptable.
Samples of the formulations prepared for this study on week 1 and week 2 were analysed to check the homogeneity and concentration.
Chemical analysis were carried out by the Analytical Chemistry Department of RTC. Results of the analyses were within the limits of
acceptance stated in the RTC SOPs for suspensions (90-110% for concentration and CV
<10% for homogeneity).
Details on mating procedure:
The females will be paired with male rats. Females will be paired one to one in the home
cage of the male and left overnight. The day of mating, as judged by the presence of sperm
in the vaginal smear or by the presence of a copulation plug, will be considered as Day 0 of
gestation (or Day 0 post coitum). Full mating records will be maintained.
Duration of treatment / exposure:
days 6 to 19 post coitum
Frequency of treatment:
once daily
Duration of test:
up to day 20 of gestation
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
nominal conc.
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were in agreement with the Sponsor based on a previous study in rats
Maternal examinations:
- Time schedule: each animal was observed daily, for mortality all animals were checked early in the morning and again in the afternoon, at weeends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day

- Time schedule: during the dosing period an additional observation for signs of reaction to treatment was performed at approx. 1,5-2 h after dosing

- Time schedule for examinations: all animals were weighed an Days 0, 6, 9, 12, 15 and 20 post coitum

FOOD CONSUMPTION: Yes, food consumption was measured on Days 6, 9, 12, 15 and 20 post coitum starting from Day 0 post coitum

- Sacrifice on gestation day 20
- Organs examined: necropsy (including examination of the external surface and orifices)

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number, sex and weight of all live foetuses, number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing), gross evaluation of placentae, uteri or individual uterine horns without visible implantations were immersed in a 20 % solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation
Fetal examinations:
- External examinations: Yes: all live foetuses
- Soft tissue examinations: Yes: approx. half per litter
- Skeletal examinations: Yes: approx. half per litter
- Head examinations: No
For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Pre-implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of implantations) x 100/no. of corpora lutea
Post-implantation loss was calculated as a percentage from the formula: (no. of implantations - no. of live young) x 100/no. of implantations
Total implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of live young) x 100/no. of corpora lutea
Sex ratios of the foetuses were calculated as the percentage of males per litter
All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the litter percentage of affected litters relative to all litters.
Historical control data:
no historical data were usesd for comparison with test data
Details on maternal toxic effects:
Maternal toxic effects: yes

Details on maternal toxic effects:
Mortality and fate of animals: No mortality occurred during the study. A total of 4 females were found not pregnant at necropsy: one in the control and mid-
dose groups and two in the low dose group. Total resorption was detected in two low dose females. In our experience both incidences are considered within a normal variability.
The number of females with live foetuses on gestation Day 20 was 23 in the control and mid-dose groups, 20 in the low dose group and 24 in the high dose

Clinical signs: at 1000 mg/kg/day increase of staining of different regions of the body surface, mainly noted on head areas (14 out of 24 females), soft faeces.

Body weight performance: a statistically significant decrease in body weight (approximately from 4% on gestation Day 12 to 17% on gestation Day 20) and body weight gain (approximately from 34% on gestation Day 9 to 54% on gestation Day 20) was found in females at 1000 mg/kg/day, when compared to controls,
indicating a condition of maternal toxicity.

Food consumption: a statistically significant decrease in food consumption was found at 1000 mg/kg/day, when compared to controls, starting from Day 9
post coitum and reaching a decrease of approximately 37% on Day 20 post coitum

Terminal body weight, uterus weight and absolute weight gain: statistically significant reductions in terminal body weight and gravid uterus weight (of
approximately 16%) and absolute weight gain (of approximately 67%) were evident at 1000 mg/kg/day when compared to controls

Macroscopic observations: at post mortem examination was found staining of different regions of the head in 8 out of 24 females at 1000 mg/kg/day group and hairloss of different regions of the body surface in 4 out of 24 females of the same group.
Key result
Dose descriptor:
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity:
Key result
Dose descriptor:
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity:
Fetal body weight changes:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Foetal weight: foetal weight and consequently litter weight at 1000 mg/kg/day were statistically significantly lower than control (-12 %)
No relevant differences were seen in the other litter data parameters investigated.

Foetal abnormalities and variants: a marked increase in small foetuses (weight less than 2.70 g) was noted at 1000 mg/kg/day compared with all other groups (2 in the control group, 3 in the low dose group, 5 in the mid-dose group and 86 in the high dose group), this finding could be considered as a consequence of the maternal toxicity noted in this group.
Malformations such as astomia and agnatia were detected in one foetus in the mid-dose group (dam no. 115 foetus no. 10) and were confirmed at skeletal examination of the foetus, the anomaly of forepaw detected in one high dose foetus was confirmed as malformation at skeletal examination, the incidence of these major abnormalities are considered within a normal variability.
Malformations such as absence of skull bones, pubis no ossification and malpositioned bones were detected in two mid-dose foetuses and two high dose foetuses, an increased incidence in incomplete or no ossification of most parts of the skeleton was noted in high dose foetuses compared to control foetuses, most of these changes were considered correlated to the lower foetal weight associated with maternal toxicity noted in this group
malformations such as extremely enlarged ureter associated with extreme pelvic dilatation of kidneys were noted in one mid- and one high dose foetus, extremely enlarged ureter was also detected in two control foetuses, because the incidence of these malformations was low and not dose-related, they were considered to be incidental.
Key result
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
not specified
Key result
Developmental effects observed:
not specified
At the dosage of 1000 mg/kg/day, the source substance caused a marked maternal toxicity as indicated by the reduction in food consumption, body weight, body weight gain, gravid uterus weight and absolute weight gain. As a consequence of the marked maternal toxicity, at the same dosage, foetal toxicity was present as demonstrated by the reduction in litter weight, foetal weight and delay in the ossification of different parts of the foetal skeleton.
Visceral malformations detected in mid- and high dose groups were considered common findings in foetuses, that usually disappear shortly after birth and since the incidence was low and not dose-related, they did not show clear relationship to treatment and therefore were considered incidental.
Skeletal findings detected in the same groups were considered to be a consequence of the low foetal weight and due to the signs of maternal toxicity observed in the high dose group, rather than to a direct effect of the test item on foetuses.
No signs of maternal and foetal toxicity were noted at the dosages of 100 and 300 mg/kg/day.
On the basis of the results obtained in this study, the dosage of 300 mg/kg/day could be considered the NOAEL for maternal toxicity and the dosage of 1000 mg/kg/day the NOAEL for embryo-foetal effects. No exposure related developmental toxic effects were observed in the study.
Executive summary:

The source substance 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester was tested in rats to detect the effects on pregnant animals, when the material was administered during the period of organogenesis. The study design was based on OECD Guideline of Testing of Chemicals No. 414 "Prenatal" Developmental Toxicity Study", adopted 22nd January 2001.

Mated female Sprague Dawley rats were randomised into 3 test groups and a control group, each containing 24 animals. The animals were dosed by oral gavage over Days 6 -19 of gestation. Dose levels administered were 0, 100, 300 and 1000 mg/kg/day.

Animals were regularly monitored during gestation for clinical signs of toxicity and for effects on body weight and food consumption performance. They were killed on Day 20 of gestation. The status of each implantation was recorded and the foetuses were weighed and examined for visceral and skeletal abnormalities, including the state of skeletal ossification.

On the basis of the results obtained in this study, the dosage of 300 mg/kg/day could be considered the NOAEL for maternal toxicity and the dosage of 1000 mg/kg/day the NOAEL for embryo-foetal effects. No exposure related developmental toxic effects were observed in the study.

Data source

Materials and methods

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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