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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
November to December 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
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): [trade name]
- Substance type: product
- Physical state: colourless liquid
- Composition of test material, percentage of components: esters of 1,2,4-benzenetricarboxylic acid with linear C8- and C10-alcohols, 40-60 % C8 and 40-60% C10
- Stability under test conditions: the stability at 24 hours at room temperature was
verified
- Storage condition of test material: at room temperature

For historical reasons another CAS-number/EINECS-name is used for the same substance: 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (EC Number 268-007-3, CAS Number 67989-23-5). Both EC-numbers/CAS-numbers refer to the identical substance.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- 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

ENVIRONMENTAL CONDITIONS
- 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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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.

VEHICLE
- 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:
yes
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
No. of animals per sex per dose:
24
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

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- 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

DETAILED CLINICAL OBSERVATIONS: Yes
- 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


BODY WEIGHT: Yes
- 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

POST-MORTEM EXAMINATIONS: Yes
- 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
Statistics:
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.
Indices:
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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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
group.

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.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects of toxicological concern

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion