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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Materials, methods and results well described/presented in text and tables
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
Assessment of the development toxicity of 2-Ethylhexanoic acid in rats and rabbits
Hendrickx, A. G. et al.
Bibliographic source:
Fundamental and Applied Toxicology 20: 199 - 209.
Reference Type:
Developmental toxicity of 2-ethylhexanoic acid (2-EHA) by gavage in Fischer 344 rats and New Zealand White rabbits
Fisher, al.
Bibliographic source:
The Toxicologist 9: 269.
Reference Type:
TOXICOLOGICAL EVALUATION No. 275 2-ethylhexanoic acid 06/00
BG Chemie
Bibliographic source:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The study investigated the developmental toxicity of 2-ethylhexanoic acid (2-EHA) in New Zealand White rabbits at the dose levels of 25, 125, and 250 mg/kg/day given by gavage. The treatment period with the test substance lasted from the gestational day 6 to the gestational day 18. The rabbits were sacrificed on gestational day 29. A control group receiving the vehicle was also used. The following examinations were carried out on the dams: clinical signs, morbidity, mortality, body weight, food consumption, reproductive organs, abdominal and thoracic cavities (grossly), corpora lutea, gravid uterus weights, number of dead and live foetuses, resorptions, liver weight. Lastly, the following foetal examinations were carried out: weight, sex, external malformations, visceral and skeletal evaluations.
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexanoic acid
EC Number:
EC Name:
2-ethylhexanoic acid
Cas Number:
Molecular formula:
2-ethylhexanoic acid
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): 2-EHA (butylethylacetic acid, Union Carbide Corp., Texas City, TX)
- Analytical purity: 99.4%

Test animals

New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Virgin New Zealand white rabbits
- Source: Hazleton-Dutchland Laboratories, Inc. (Denver, PA)
- Weight: 2.5 - 3.5 kg
- Housing: animals were singly housed in stainless-steel wire-mesh cages.
- Diet (ad libitum): Agway Prolab Certified Chow, Agway, Inc.
- Water (ad libitum): water (Municipal Authority of Westmoreland County)
- Quarantine period: approximately 2 weeks

Animals were screened for intestinal parasites prior to assignment to the study.

- Temperature (°C): 18.9 - 20.6 °C
- Relative humidity: 40 - 60%
- 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.
A dose volume of 2 mL/kg was employed based on the body weight of each animal on gestational day 6.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Dosing concentrations were prepared and analysed for stability, homogeneity, and concentration prior to the onset of the study. The analyses were done by means of a Hewlett-Packard 5880 gas chromatograph (GC) equipped with a flame ionization detector and a 30-mm DB-1 fused silica capillary column with 1.0 µm film thickness. The carrier gas was ultrahigh-purity helium, and the column flow rate was approximately 2.0 mL/min. A 1.0-µL aliquot of dosing solution, diluted with propanol, was injected into the GC for analysis. Standard ranged from 50 to 150 ng 2-EHA/mL in propanol.
The results indicated that 2-EHA was distributed uniformly in corn oil and remained stable for at least 21 days when stored at room temperature. Based on these results, the dosing formulations were prepared once and analyzed prior to the onset of the dosing period. Concentration verification showed analytical values ranging from 92.5 to 109.7% of the nominal dosing concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: female rabbits (at least 2.5 kg) were naturally bred (one male:one female) to proven fertile males, and the day of observed mating was designated gestational day 0 (Hafez, 1070)*. Males from the Bushy Run Research Center rabbit breeding colony were used to generate timed-pregnant females.

- Hafez, E.S.E. (1970). Reproduction and Breeding Techniques for Laboratory Animals Lea and Febiger, Philadelphia.
Duration of treatment / exposure:
Gestational day 6 - 18
Frequency of treatment:
Duration of test:
29 days
No. of animals per sex per dose:
15 mated female rabbits
Control animals:
yes, concurrent vehicle
Details on study design:
- Animal assignment: fifteen mated female rabbits were randomly assigned to each treatment group such that all groups were equivalent in mean body weight and body weight range on gestational day 0. Eight animals per treatment group were similarly assigned to the range-finding study.

- Range-finding study: pregnant rabbits were orally dosed on gestational days 6 - 18 with 125, 250, 500, or 1000 mg 2-EHA/kg/day in corn oil in the range-finding study.
Results range-finding study:

- no treatment-related effects on corrected (minus gravid uterus) weights, gestational weight changes, or liver weights were observed at necropsy on gestational day 29.
- no developmental toxicity, as judged by resorptions and dead or malformed foetuses, was observed.

1000 mg 2-EHA/kg/day
- treatment with the dose level was highly toxic to pregnant rabbits; all eight (100%) does died during the dosing period
- clinical signs included hypoactivity, laboured respiration, and ataxia

500 mg 2-EHA/kg/day:
- treatment with the dose level was highly toxic to pregnant rabbits; seven of eight (87.5%) does died during the dosing period
- increase incidence of hypoactivity was seen

250 mg 2-EHA/kg/day:
- one maternal death occurred during or after treatment
- one female aborted on gestational day 25
- increase incidence of hypoactivity was seen

125 mg 2-EHA/kg/day:
- one maternal death occurred during or after treatment
- one female aborted on gestational day 25
- a decrease in mean foetal body weights which was most probably due to the increase in the litter size (11.3 foetuses/litter) in this group relative to that in the control group (7.0 foetuses/litter)(Tyl, 1987)*

Vehicle control group:
- one maternal death occurred during or after treatment

* Reference:
Tyl, R.W. (1987). Developmental toxicity in toxicology research and testing. In Perspectives in BAsic and Applied Toxicology (B. Ballantyne, Ed.), pp. 203 - 238. Wright/Year Book, Boston/London.


Maternal examinations:
- Time schedule: all females were examined twice daily
- Cage side observations checked: clinical signs of toxicity, morbidity, and mortality


- Time schedule for examinations: gestational days 0, 6 (for dose calculations), 9, 12, 15, 18, and 29

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
Food consumption was measured daily on gestational day 0 - 29.


- Sacrifice on gestation day 29 by intravenous injection of T-61 solution (Hoechst Corp., Somerville, NJ) into the marginal ear vein.
- Organs examined: reproductive organs
All maternal livers were weighed; liver sections were fixed in 10% buffered formalin in the definitive study.
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: No data
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Apparent nongravid uteri were placed in 10% ammonum sulfide solution for detection of early resorptions (Salewski, 1964)*.

* Reference:
- Salewski, E. (1964). Farbemethode zum makroskopischen nachweis von implantations-stellen am uterus der ratte. Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol. 247, 367.
Fetal examinations:
- The number of live and dead foetuses were recorded.
- Viable foetuses were weighed and their sex was determined
- Viable foetuses, which were killed by intraperitoneal injection of sodium pentobarbital
- External examinations: Yes, all foetuses
- Soft tissue examinations: Yes, all foetuses were inspected for visceral abnormalities by modification of methods described by Staples (1974)*. Approximately 50% of each litter was examined for soft tissue craniofacial malformations/variations.
- Head examinations: Yes, the heads of foetuses were fixed in Bouin's solution and analysed by sectioning methods modified from Wilson (1965, 1973)* and van Julsingha and Bennett (1977)*
- Skeletal examinations: Yes, all foetuses were eviscerated, fixed in ethanol, and processed with alizarin red S (Crary, 1962; Peltzer and Schardein, 1966)* for skeletal examination.

* References:
- Staples, R.E. (1974). Detection of visceral alterations in mammalian fetuses. Teratology 9: A-37.
- Wilson, J.G. (1965). Embryological considerations in teratology. In Teratology Prinicples and Techniques (J.G. Wilson and J. Warkany, Eds.), pp. 251 - 277, Univ. of Chicago Press, Chicago/London.
- Wilson, J.G. (1973). Environment and Birth Defects. Academic Press. New York.
- Crary, K.D. (1962). Modified benzyl alcohol clearing of alizarin-stained specimens without loss of flexibility. Stain Technol. 37, 124 - 125.
- Peltzer, M.A., and Schardein, J.L. (1966). A convenient method for processing foetuses for skeletal staining. Stain Technol. 41, 300 - 302.
The unit of comparison was the pregnant female or the litter (Weil, 1970)*. Quantitative continuous variables (e.g., weights and measurements) were statistically analysed by means of Levene's test for equal variances (Levene, 1960)*, analysis of variance (ANOVA), and t tests with Bonferroni probabilites for pairwise comparisons. For homogeneous variances, the pooled t test was used when the ANOVA was significant; for heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Brown and Forsythe, 1974)* and separate variance t tests. Nonparametric data (e.g., number of live and dead foetuses) were analysed using the Kruskal-Wallis test followed by the Mann-Whitney U test (Sokal and Rohlf, 1969)* when appropriate. The litter incidence of malformed foetuses and foetsus with variations was compared using Fisher's exact test (Sokal and Rohlf, 1969)*.

* References:
- Weil, C.S. (1970). Selection of the valid number of sampling units and a consideration of their combination in toxicological studies involving reproduction, teratogenesis or carcinogenesis. Fed. Cosmet. Toxicol. 8, 177 - 182.
- Levene, H. (1960). Robust tests for equality of variance. In Constributions to Probability and Statistics (I. Olkin et al., Eds.), pp 278 - 292. Stanford Univ. Press, Stanford, CA.
- Brown, M.B., and Forsythe, A.B. (1974). The small sample behavior of some statistics which test the equality of several means. Technometrics 16, 12 9 - 132.
- Sokal, R.R., and Rohlf, F.J. (1969). Biometry. Freeman, San Francisco.
no data
Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- no treatment-related gross pathological findings or differences in corrected (minus gravid uterus) weight or gestational weight change (gestational day
29 - gestational day 0) or absolute or relative liver weight at necropsy on gestational day 29.

250 mg/kg/day:
- one pregnant female died at gestational day 16
- reduction in maternal body weight change ( p<0.01) and food consumption was observed during the post-treatment period (gestational day 18 - 29)

125 mg/kg/day:
- one pregnant female died at gestational day 15
- one doe aborted on gestational day 27

Effect levels (maternal animals)

Dose descriptor:
Effect level:
25 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:
- no effect of treatment on prenatal mortality (resorptions and dead foetuses) or the percentage of viable foetuses.
- foetal body weights (both sexes) and the sex ratio (percentage males) were equivalent for all groups.
- no differences in the incidence of external, visceral, or skeletal malformations or variations among all groups; dilated lateral and third ventricles with no tissue compression were observed in all groups, but differences were not significant.

Effect levels (fetuses)

Dose descriptor:
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

According to the authors of the publication, the study revealed no teratogenic effects in New Zealand White rabbits following exposure to doses of 2-EHA during organogenesis. Maternal toxicity occurred in the absence of developmental toxicity. Thus, the NOAEL for adverse developmental effects in rabbits is at least 125 mg/kg/day. Based on maternal death and the abortion at 125 mg/kg/day, the corresponding maternal NOAEL is 25 mg/kg/day.