Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
exposure duration was only from day 6-15 of gestation instead of GD 19;
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, C16-18, 2-ethylhexyl esters
EC Number:
292-951-5
EC Name:
Fatty acids, C16-18, 2-ethylhexyl esters
Cas Number:
91031-48-0
Molecular formula:
C24H48O2 to C26H52O2
IUPAC Name:
91031-48-0
Details on test material:
- Name of test material (as cited in study report): 2-ethylhexylstearate
- Physical state: colourless and odourless liquid
- Analytical purity: 100 %
- Lot/batch No.: 1006
- Expiration date of the lot/batch: Oct 1993
- Stability under test conditions: stable in arachidis oil, DAB 10
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: approx. 8-10 weeks
- Weight at study initiation: mean approx. 197 g
- Housing: individually in Makrolon Type M3 cages (Ebeco) with standard softwood bedding (ARWI-Center, Essen, Germany)
- Diet: Pelleted Altromin Maintenance Diet 1324, Lot No. 221092/1558 (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-23 °C
- Humidity: 40-56 %
- Air changes: 10-15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Arachidis oil, DAB 10
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility of test substance
- Concentration in vehicle: 20, 60 and 200mg/mL
- Amount of vehicle (if gavage): 5mL/kg bw

After delivery all females were assigned to the different groups using a computer-generated random algorithm.

The test substance was administered orally by gavage once daily in the morning from day 6 up to day 15 post coitum inclusive. All groups received a dose volume of 5 mL/kg body weight, adjusted to the body weight of day 6 post coitum. Control animals were similarly dosed with the vehicle alone.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant, at day 0
- Proof of pregnancy: vaginal plug day 0 of pregnancy
The females were mated at the supplier with an accurate day of mating. They were received at the testing facility on day 0 of gestation.
Duration of treatment / exposure:
from day 6 up to day 15 of gestation
Frequency of treatment:
once daily
Duration of test:
until day 20 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (group 1), 100 (group 2), 300 (group 3) and 1000 (group 4) mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
Mortality rate: The animals were checked at least twice daily for any mortalities.
Any female sacrificed or found dead during the study was subjected to macroscopic examination of the visceral organs, with emphasis on the uterus and its content.
Signs and/or symptoms: The animals were observed at least twice daily (working days) for signs of reaction to treatment and/or symptoms of illness.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Post mortem examination, including gross macroscopic examination of all maternal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea, was performed and the data recorded.

BODY WEIGHT: Yes, mean body weight changes
- Time schedule for examinations: days 0, 6, 16 and 20 of gestation
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: dead/living foetuses

Number and distribution of intrauterine implantations were classified as live or death fetuses, late intrauterine deaths (resorptions), early intrauterine (resorption sites). The fetuses were removed from the uterus. Intrauterine deaths were classified on the basis of the presence (late) or absence (early) of fetal or decidual tissue in addition to placental tissue.
Fetal examinations:
- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: half per litter: malformations oh hydrocephalus, variations of brain, adrenal gland, renal pelvis, ureter
- Skeletal examinations: Yes: half per litter: malformations of hydrops, retardations of skull bones, hyoid, sternebrae, pelvis, 13th rib
- Head examinations: Yes: half per litter

The live fetuses were sexed, weighed individually including placentae, examined for gross external abnormalities and allocated to one of the following procedures:
1) Half of the fetuses from each litter was non individually fixed in Bouin's solution in order to examine viscera and brain by Wilson's slicing technique. After examination the sections were not preserved.
2) The remaining fetuses were placed non individually in a solution of potassium hydroxide for clearing and were stained with alizarin red (Shandon Varistain 24-T). The skeletons were examined and preserved in plastic containers. All abnormalities were recorded.
The uteri (including content) of all females were weighed at necropsy on day 20 post coitum to enable the calculation of the corrected body weight gain.
Statistics:
The following statistical methods were used:
If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on a pooled variance, was applied for the comparison between the treated groups and the control group.
The Steel-Test was applied when the data could not be assumed to follow a normal distrubution.
Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).
Historical control data:
Findings both on the individual foetus and an the litter basis did not differ from the historical control obtained in six developmental toxicity studies on the same species.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
The dams tolerated the applied dose levels of up to 1000mg/kg bw/day without lethality and clinical signs of systemic toxicity.
Maternal body weight gain was not affected by the treatment.

Mortality:
No death occurred in the dams of group 1 (vehicle control) and in the test groups 2 - 4.

Signs and/or symptoms:
No compound-related symptoms were observed in all treatment groups. In one female (group 3) was noted a skin incrustion on the back and another female (group 1) was severely aggressive by handling.

Body weight gains and corrected body weight:
Body weight profiles of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of the treatment groups compared favourably with the control values.

Reproduction data:
No compound-related differences were noted between the mean reproduction data of the test groups in comparison to the control group. In the group 2 and 4 the post-implantation loss and total embryonic deaths were significantly decreased. These findings were considered to be incidental because of the high control values. Furthermore the number of total fetuses was increased in the group 2 and 4, which is also incidental because there was no dose-relationship.

Necropsy findings:
No macroscopic changes were noted in the dams of the groups 1 - 4.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Apart from dose group 1000 mg/kg bw/day (one dead foetus) all females had viable foetuses. Pre- and postimplantation loss and mean numbers of resorption were unaffected by treatment. All parameters were comparable with the animals of the control group. Skeletal and visceral investigations detected no treatment-related malformations.

Body weight:
The weights of live fetuses exibited no significant differences on a litter and individual basis e.g. mean weight between the control group and the treatment groups.

Placenta and uterus weight:
The weights of placentae and the whole uterus showed no significant differences between the control group and the treatment groups.

Sex ratios:
The sex ratio of the fetuses was not effected by the treatment with the test substance.

External examinations:
No macroscopical findings were noted at external examination of fetuses which were considered to be an effect of the treatment with the test article. In the group 1 was noted a beginning hydrops and in the group 4 one fetus with paleness and one dead fetus.

Visceral examination:
The findings were as follows:
Group 1: 127 examined fetuses
28 hydronephrosis
9 ureter dilatation
5 ureter waved
1 runt, brain lateral sinus dilatation, other organs normal
1 thorax - blood coagulum [artifact]
1 adrenal central pinhead cyst [suspicious]
1 umbilical region - gut protrusion [artifact]

Group 2: 138 examined fetuses
34 hydronephrosis
12 ureter dilatation
3 ureter waved
1 runt, hydrocephalus internus

Group 3: 138 examined fetuses
26 hydronephrosis
5 ureter dilatation
6 ureter waved
1 ear region subcutaneous hematoma
1 umbilical region - gut protrusion [artifact]

Group 4: 140 examined fetuses
24 hydronephrosis
10 ureter dilatation
8 ureter waved
1 inguinal hernia, protrusion of gut and testis between peritoneum and trunk, muscles [artifact]
1 runt, brain lateral sinus dilatation, other organs normal

The visceral examination of the preserved fetuses did not reveal any treatment-related abnormalities.

Skeletal examination of fetuses:
Retardations:
Group 1: 141 examined fetuses
Group 2: 152 examined fetuses
single sternebrae non ossified,
significant increase at level 1% (34 fetuses out of 22 dams)
Group 3: 150 examined fetuses: no significant findings
Group 4: 152 examined fetuses
single sternebrae non ossified,
significant increase at level 5 % (29 fetuses out of 22 dams)
two sternebrae non ossified,
significant increase at level 1 % (21 fetuses out of 22 dams)

The statistically significant differences were considered to be incidental because these retardation effects were not accompanied by weight retardation of the treatment groups. The incidental character of these retardations is emphasized by the fact the values were within the normal range of variation for this strain.

Variations (examined fetuses):
Group 1: no variations
Group 2: no variations
Group 3: no variations
Group 4: no variations

Malformations (examined fetuses):
Group 1: 1 fetus beginning hydrops
Group 2: no findings
Group 3: no findings
Group 4: no findings


Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

2 -ethylhexyl stearate up to a dose of 1000 mg/kg bw/day does not produce any embryo- and foetotoxic or teratogenic effects. The NOAEL for maternal-, developmental-, embryo-, foetotoxicity and teratogenicity is deduced 1000mg/kg bw/day.

2 -ethylhexyl stearate has not to be classified according to DSD and CLP.

Applicant's summary and conclusion