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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, with minor restrictions in design and/or reporting but otherwise adequate for assessment.

Data source

Referenceopen allclose all

Reference Type:
study report
Report Date:
Reference Type:
other: EPA internal memorandum
Report Date:

Materials and methods

Test guideline
according to
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
GLP compliance:
Limit test:

Test material

Details on test material:
Purity: 99.4% (Union Carbide Corp.)

Test animals

Fischer 344
Details on test animals and environmental conditions:
- Source: Charles River
- Age at arrival: males: 70 days; females: 63 days
- Weight at study initiation: males: 175-200 g; females: 130-150 g
- Housing: stainless steel wire mesh cages
- Diet (e.g. ad libitum): Prolab Certified Ground Rodent Chow, RMH-3200; ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

- Temperature (°F): 69-72
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:
The test chemical was diluted in certified corn oil (Mazola, Best Foods, Inc., CPC International) and mixed by inversion prior to the onset of the study. GC analysis indicated that the test material was stable for at least 21 days when stored at room temperature and uniformly distributed in corn oil. A dose volume of 2 ml/kg bodyweight (concentration in vehicle: 0 - 250 mg/ml) was administered by gavage, based on the body weight of each animal on gestation day 6.

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Actual concentrations of test material in corn oil were determined by GC analysis and were 50.4, 132.0 and 246.0 mg/ml for the 100, 250 and 500 mg/kg/day doses, respectively. Analysis values ranged from 92.5 to 109.7% of the nominal dosing concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Each male was used only once
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy (GD 0)
- Successfully mated females weighing 147-174 g were housed singly and were randomly assigned (stratified by body weight) to each experimental group.
Duration of treatment / exposure:
Gestation day 6 through 15
Frequency of treatment:
Duration of test:
Up to gestation day 21
Doses / concentrations
Doses / Concentrations:
0, 100, 250 or 500 mg/kg bodyweight/day
nominal in diet
No. of animals per sex per dose:
25 (females only)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose range finding study


Maternal examinations:
- Time schedule: daily

- Time schedule for examinations: on GD 0, 6 (prior to dosing), 12, 15, 18 and 21.

- Food consumption (g food/animal/day) was measured for the intervals GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21.

- Sacrifice on gestation day 21 (CO2)
- Gross examination: gravid uterus, ovaries (including corpora lutea), cervix, vagina and abdominal and thoracic cavities.
- Organ weights: liver, uterus
- The liver was fixed for possible future examination
Ovaries and uterine content:
The uterus was externally examined for signs of hemorrhage, removed from the abdominal cavity and dissected longitudinally to expose the contents. All live and dead fetuses and resorption sites were noted and recorded. Uteri from females that appeared nongravid were placed in ammonium sulfide for detection of early resorptions.
Fetal examinations:
All live fetuses were weighed and sexed. All fetuses were examined for external malformations including cleft palate and variations. One half of the fetuses in each litter were examined for thoracic and abdominal visceral abnormalities. These fetuses were decapitated and their heads were fixed for examination of craniofacial structures. Remaining fetuses were eviscerated, fixed and examined for skeletal malformations and variations. Decapitated fetuses were also processed for staining but were not examined.
The unit of comparison was the pregnant female or the litter. Results of the quantitative continuous variables were intercompared for the dose groups by the Levene's test for equal variances, ANOVA, and t-tests with Bonferroni probabilities for pairwise comparisons. When Levene's test indicated homogeneous variances and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by ANOVA for unequal variances followed, when necessary, by the separate variance t-test. Nonparametric data obtained following laparohysterectomy were treated using the Kruskal-Wallis test followed by the Mann-Whitney U test when appropriate. Incidence data were compared using Fisher's Exact Test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: hypoactivity, ataxia, ocular discharge

Details on maternal toxic effects:
Clinical signs were observed at the high-dose level only and included hypoactivity, ataxia, audible respiration, ocular discharge and periocular encrustations. No effects on body weight and mortality were observed. Liver weight (absolute and relative) was increased in the high-dose group.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
250 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
100 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: reduced skeletal ossification

Details on embryotoxic / teratogenic effects:
Fetal body weights per litter were reduced in the high-dose group, but these findings may be confounded by the slightly larger mean litter size in this group. Reduced skeletal ossification was observed in the mid- and high-dose group. Dilation of the lateral ventricles of the brain with no tissue compression and extra (14th) thoracic centrum and arches were observed in all groups but were significantly increased at the high dose only.

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Prgenant rats were dosed with 0, 100, 250 or 500 mg/kg bw/d ethylhexanoic acid from gestation day 6 to 15. Based on the observed effects the NOAEL(maternal) was considered to be 250 mg/kg bw/d and the NOAEL(developmental) was set at 100 mg/kg bw/d.
Based on a read-across approach, the same NOAELs are taken into account in case of sodium 2-ethylhexanoate.
Executive summary:

A read across was performed from the source substance 2 -ethylhexanoic acid to the target substance sodium 2 -ethylhexanoate (for read across justification please refer to attached document, IUCLID Chapter 13).

Timed-pregnant Fischer 344 rats were exposed to 2 -ethylhexanoic acid by gavage on gestational day 6 through 15 at doses of 0, 100, 250 of 500 mg/kg bw/d in corn oil (25 plug-positive females/group). The does volume employed was 2 mL/kg bw based on the weight of each femal on gestation day 6. Clinical observations were taken daily and maternal body weights were taken on gestation day 0, 6, 12, 15, 18 and 21. Food consumption was measured for the intervals gestation days 0 -3, 3 -6, 6 -9, 9 -12, 12 -15, 15 -18 and 18 -21. At scheduled sacrifice on gestation day 21, the dams were evaluated for body weight, liver weight, gravid uterine weight and status of implantation sites. Maternal livers were retained in fixative for possible subsequent microscopic examination. Live fetuses were dissected from the uterus, counted, weighed, sexed and examined for external abnormalities. Approx. one-half of the live fetuses in each litter were examined for visceral malformations and variations. These fetuses were then decapitated and the heads feixed in Bouin`s solution; serial free hand sections of the heads were examined for soft tissue craniofacial malformations and variations. The remaining (intact) fetuses in each litter were eviscerated, fixed in alcohol, stained with alizerin red S and examined for skeletal malformations and variations.

There were no treatment-related maternal deaths. No dams aborted. One female, at 250 mg/kg bw/d, delivered early, and was removed from the study. A total of 21 -24 litters were examined in each dose group. There were no statistically significant differences among the groups for gestational maternal body weights or weight gain, or food consumption. Treatment-related clinical signs, observed only at 500 mg/kg bw/d, included hypoactivity, ataxia, and audible respiration; clinical signs with significantly increased incidence at 500 mg/kg bw/d included ocular discharge and periocular encrustation. At the gestation day 21 sacrifice there were no effects of treatment on maternal body weight or gestational weight gain, or on gravid uterine weight. Liver weights were significantly increased at 500 mg/kg bw/d. Gestational parameters, including number of ovarian corpora lutea, viable and non-viable implantations/litter, and sex ratio were unaffected by treatment. Fetal body weight/litter were significantly reduced at 500 mg/kg bw/d.

There was no significant increase in the incidence of malformations in any treatment groups relative to controls. There were no treatment-related differencesamong groups for individual external variations or for pooled external, visceral, skeletal or total variations. One visceral variation, dilated lateral ventricles on the brain with no tissue compression, exhibited an increased incidence at 500 mg/kg bw/d. Twenty-six fetal skeletal variations exhibited significantly different incidences at 500 mg/kg bw/d relative to those in controls; 19 of these were significant increases in incidence and seven were significant reductions in incidence (almost all of the reductions were in incidence of poorly ossified skeletal districts since the incidences of these same districts, unossified, increased). In addition, there were skeletal variations with increased incidences at 250 mg/kg bw/d, indicative of minimal fetotoxicity.