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Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Read-across to similar substance.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Full information on the read-across approach is provided in the attached justification document.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source and target substances are composed of the same chemical groups and are bonded together in functionally the same way. The source substance and target substances have low water solubility, high partition coefficient, and are in the physical form of a liquid with a neutral pH. Available toxicity information on the source and target substances indicates that they behave in substantially similar ways in the body. The potential for reproductive toxicity is therefore the same in all substances.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The substances are hydrophobic oils at room temperature, with no hydrophilic groups and poor water solubility (all < 0.1 mg/l). The only chemical group present in each substance is the ester group. The boiling points are > 160°C, typically with decomposition >300 °C. The substances are also lipophilic, all with partition coefficients >7.2. The relative densities are all closely related, all being approximately 0.85
All are UVCB substances – with only one part of each substance being from a UVCB source (the stearic acid component of the 2-ethylhexyl stearate, and the alcohol components of the target substances. All the carbon-carbon bonds are saturated, giving a constant ratio of one carbon to two hydrogens across all the substances.
Each substance also has a branched component, which is present from the ester due to the mono-constituent substance used to synthesise it. In the case of the 2-ethylhexyl stearate, this is the alcohol part of the substance. In the case of the target substances Isodecyl 3,5,5-trimethylhexanoate and Isotridecyl 3,5,5-trimethylhexanoate, both the acid part and the alcohol part are branched (although the alcohol is varied due to the UVCB alcohols used as an ingredient for both). The C12-15 Alkyl Ethylhexanoate has the same branched aliphatic chain that the 2-ethylhexyl stearate does, but it is from the acid component rather than the alcohol component.
A difference is that the variation for the 2-ethyl hexyl stearate is along the acid but only in increments of two carbons, which are all linear. The target substances show variation in the alcohol part of the ester, and this variation involves both linear and branched aliphatics – with the C12-15 Alkyl Ethylhexanoate showing additional variation here due to the number of carbons being between twelve and fifteen, whereas they are constant for Isodecyl 3,5,5-trimethylhexanoate (ten carbons) and Isotridecyl 3,5,5-trimethylhexanoate (thirteen carbons). The three target substances also all have a branched acid component which the source substance does not share.
The source substance also only has an even numbered amount of carbons in the acid component, whereas this is only shared in the C12-15 Alkyl Ethylhexanoate, with the Isodecyl 3,5,5-trimethylhexanoate and Isotridecyl 3,5,5-trimethylhexanoate having an odd numbered amount of carbons in the acid component.
The alcohol component of the 2-ethylhexyl stearate is both branched, which it shares with C12-15 Alkyl Ethylhexanoate, Isodecyl 3,5,5-trimethylhexanoate, and Isotridecyl 3,5,5-trimethylhexanoate. The alcohol component is also even numbered, which it only shares with C12-15 Alkyl Ethylhexanoate and Isodecyl 3,5,5-trimethylhexanoate, and not Isotridecyl 3,5,5-trimethylhexanoate. It should also be noted that due to the nature of the alcohol component of C12-15 Alkyl Ethylhexanoate, both even numbered and odd numbered amounts of carbons are present in the alcohol chain in the substance.
The substances have also been tested for toxicity (oral) and the conclusions were that neither the source substance nor target substances were classified for toxicity (LD50 > 2000 mg/kg bw). In addition, skin and irritation tests also concluded no classification under CLP 1278/2008.
These common phys-chem properties, and the similar results for toxicity and skin/eye irritation/corrosion, indicate that the substances will share similar bioavailability and toxicity.

3. ANALOGUE APPROACH JUSTIFICATION
Due to the similarities of the source and target substances with regards to branching, odd and even numbered alkyl and acid chains, the target substances are expected to behave in a substantially similar manner in vivo.
The target substances are therefore predicted to fail to induce effects of developmental toxicity in the OECD 414 study when conducted in the rat. By extension, the target substances are considered not to fulfil the criteria for reproductive toxicity under the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

4. DATA MATRIX
Full information on the read-across approach is provided in the attached justification document.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Basis for effect level:
body weight and weight gain
mortality
Abnormalities:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Abnormalities:
no effects observed
Developmental effects observed:
no

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 1981
Deviations:
yes
Remarks:
no analytical purity given
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2001
Deviations:
yes
Remarks:
Exposure duration was only from day 6-15 of gestation instead of day 5-19; no analytical purity given
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl stearate
EC Number:
244-754-0
EC Name:
2-ethylhexyl stearate
Cas Number:
22047-49-0
Molecular formula:
C26H52O2
IUPAC Name:
2-ethylhexyl stearate

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 weeks
- Weight at study initiation: mean approx. 197 g
- Housing: individually in Makrolon Type M3 cages
- Diet: Pelleted Altromin Maintenance Diet 1324 (Altromin GmbH, Lage, Germany), ad libitum (analytically controlled per batch)
- Water: tap water, ad libitum (once weekly controlled)
- Acclimation period: 5 days

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

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
- Amount of vehicle (if gavage): 5 mL/kg bw

All groups received a dose volume of 5 mL/kg body weight, adjusted to the body weight of day 6 post coitum.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant, at day 0
- Proof of pregnancy: vaginal plug day 0 of pregnancy
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 / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Other:
group 1: 0 mg/kg bw/day
group 2: 100 mg/kg bw/day
group 3: 300 mg/kg bw/day
group 4: 1000 mg/kg bw/day

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

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) The Wilson technique was applied to half of the foetuses to evaluate potential visceral changes (Wilson and Warkany, 1965).
2) The remaining fetuses were placed non individually in a solution of potassium hydroxide for clearing and were stained with alizarin red according to Dawson, 1926. All abnormalities were recorded.
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

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
The dams tolerated the applied dose levels of up to 1000 mg/kg bw/day without lethality and clinical signs of systemic toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Maternal body weight gain was not affected by the treatment. 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.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No organ weight changes were noted in the dams of the groups 1 - 4.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic changes were noted in the dams of the groups 1 - 4.
Other effects:
no effects observed
Description (incidence and severity):
Gross macroscopic examination of the maternal organs including ovaries and uterus revealed no alterations.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
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.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat. (total fraction)
Basis for effect level:
body weight and weight gain
changes in number of pregnant
clinical signs
gross pathology
mortality
organ weights and organ / body weight ratios
other: maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
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.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Apart from dose group 1000 mg/kg bw/day (one dead foetus) all females had viable foetuses. Pre- and post implantation 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.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio of the fetuses was not effected by the treatment with the test substance.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Other effects:
no effects observed
Description (incidence and severity):
The weights of placentae and the whole uterus showed no significant differences between the control group and the treatment groups.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Mean body weights and standard deviation

mg/kg Mean body weight/standard deviation
0 100 300 1000
Day 0 pc 196±13.7 198.6±17.4 197.7±18.1 195.9±20.4
Day 6 pc 235.5±19.1 236.4±17.6 237.7±18.3 232.6±20.4
Day 16 pc 308.3±29.8 309.1±25.0 313.5±22.4 309.7±27.7
Day 20 pc 362.6±40.2 361.7±28.1 370.7±29.6 364.7±30.5

Summary of the reproduction data: average per group ± standard deviation

Parameter Control 100 mg/kg 300 mg/kg 1000 mg/kg
No. of females 24 24 24 24
Pregnant females 22 22 23 23
Total pups 268 290 288 292
Corpora lutea 15.8±2.2 16.4±2.1 15.9±1.9 16.1±2.4
Implantation sites 13.8±2.7 13.6±1.8 13.6±2.8 13.5±1.9
Pre-implantation loss in % of corpora lutea 15.5 16.7 14.8 15.8
Post-implantation loss 8.8 3.3* 7.7 2.0**
• early resporptions 7.5 0.3* 7.7 1.3
• late resorptions 1.4 0 0.6 0.3
• dead foetuses 0 0 0 0.3
in % of implantation sites        
Living foetuses 12.2±3.0 13.2±1.4 12.5±3.4 13.3±1.9
Sex ratio 0.474 0.479 0.497 0.544
Foetal weight        
• male 4.3±0.7 4.1±0.5 4.5±0.9 4.3±0.9
• female 4.1±0.6 3.8±0.4 4.2±0.9 4.1±0.8
• runts (male and female1) 1.0±0.0 1.0±0.0 0.0±0.0 1.7±1.2
Placental weight 0.6±0.0 0.6±0.1 0.6±0.1 0.6±0.1

1Runts are small living foetuses exhibiting a body weight less than 75% of the mean litter weight.

*Signicantly different from control at P E0.05.

**Signicantly different from control at P E0.01.

Visceral examination - foetal incidence (f) / litter (l)

  Historical data (total) n=6 studies Historical data (A. oil) n=4 studies 2-Ethylhexyl stearate, dose level (mg/kg/day)
0 100 300 100
f l f l f l f l
No. of foetuses examined 858 142 595 97 127 22 138 22 138 23 140 22
Malformations
Hydrocephalus internus 3 3 1 1 0 0 1 1 0 0 0 0
Variations
Brain lateral sinus dilation 1 1 1 1 1 1 0 0 0 0 1 1
Adrenal gland cyst 1 1 1 1 1 1 0 0 0 0 0 0
Hydronephrosis (renal pelvis dilated) 163 69 119 52 28 15 34 13 26 15 24 13
Ureter dilated 44 27 30 18 9 6 12 8 5 5 10 5
Ureter waved 32 22 32 22 5 4 3 3 6 4 8 6

Historical vehicle data (total): 2 studies treated with water and four studies treated with arachidis oil.

Historical vehicle data (A. oil): Treated with arachidis oil.

Skeletal examination - foetal incidence (f) / litter incidence (l)

  Historical data (total) n=6 studies Historical data (A. oil) n=4 studies 2-Ethylhexyl stearate, dose level (mg/kg/day)
0 100 300 100
f l f l f l f l
No. of foetuses examined 935 142 650 97 141 22 152 22 150 23 152 22
Malformations
Hydrops 1 1 1 1 1 1 0 0 0 0 0 0
Retardations
Skull bones: incompl. oss.  37 27 22 14 3 3 4 4 2 1 3 3
Hyoid: incompl. oss.  16 12 10 8 2 2 5 4 4 3 2 2
Hyoid: non-ossified  36 22 24 12 0 0 1 1 0 0 1 1
Single sternebrae: incompl. oss. 343 118 242 82 52 18 61 22 44 17 38 15
Two sternebrae: incompl. oss. 103 59 84 45 11 8 34** 15 24 11 29* 13
Two sternebrae: non-oss.  46 24 24 16 4 2 4 4 7 5 21** 10
Several sternebrae: incompl. oss. 9 8 8 7 2 2 0 0 1 1 1 1
Several sternebrae: non-oss.  17 5 3 3 2 2 1 1 0 0 0 0
Pelvis: incompl. oss.  6 2 1 1 1 1 1 1 0 0 0 0
13th rib: incompl. oss./absent ul  9 8 7 6 0 0 2 2 3 3 2 1
Variations
Ribs additional: rudim./short ul  15 13 14 12 3 2 3 3 8 6 10 9
Ribs additional: rudim./short bl  19 15 19 15 7 6 2 2 5 4 7 5
Vertebrae: dumbbell shaped 244 97 202 74 53 19 62 20 52 17 57 20
Vertebrae: bipartited  21 20 18 17 6 6 4 4 4 4 3 3

Fisher's exact test (two-sided) signicant at level greater than P = 0.05 * or 0.01 ** (performed at foetal incidences only) (Bonferroni-Holm corrected).

Applicant's summary and conclusion

Conclusions:
The developmental NOAEL of the test material was considered to be 1000 mg/kg bw/day under the conditions of the test.
Executive summary:

2-Ethylhexyl stearate was investigated in an embryo-/foetotoxicity and teratogenicity study on rats according to OECD guidelines for the testing of chemicals (No. 414). Dose levels of 0 (arachidis oil), 100, 300 and 1000 mg/kg body weight/day were administered by gavage. Dams tolerated the applied dose levels without any toxic effects. Pre- and post-implantation loss and mean numbers of resorptions were unaffected by treatment. All parameters were comparable with the animals of the control group. Skeletal and visceral investigations revealed no treatment-related malformations. For embryo-/foetotoxicity, teratogenicity and maternal toxicity a NOAEL of 1000 mg/kg was deduced.