Hexanoic acid, 2-ethyl-, C12-15-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The OECD 421 study is not required to be conducted as a reliable OECD 414 study is available.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study waived due to provisions of other regulation

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Study conducted to recognised testing guideline with GLP conditions.

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

read-across: supporting information

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

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

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

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

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.

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.

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.

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

Abnormalities:

no effects observed

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.

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

Abnormalities:

not specified

Developmental effects observed:

not specified

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 female^1)	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).

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the result of the key study the registered substance does not meet the criteria for classification as a reproductive toxic substance under the EU classification, Labelling, and Packaging (CLP) regulation (1272/2008).