Potassium 2-ethylhexanoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated 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:

other: Materials, methods and results well described/presented in text and tables

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Assessment of the development toxicity of 2-Ethylhexanoic acid in rats and rabbits

Author:

Hendrickx, A. G. et al.

Year:

1993

Bibliographic source:

Fundamental and Applied Toxicology 20: 199 - 209.

Reference 2

Reference Type:

publication

Title:

Developmental toxicity of 2-ethylhexanoic acid (2-EHA) by gavage in Fischer 344 rats and New Zealand White rabbits

Author:

Fisher, L.C.et al.

Year:

1989

Bibliographic source:

The Toxicologist 9: 269.

Reference 3

Reference Type:

publication

Title:

TOXICOLOGICAL EVALUATION No. 275 2-ethylhexanoic acid 06/00

Author:

BG Chemie

Year:

2000

Bibliographic source:

BG Chemie InfoCenter TOXIKOLOGISCHE BEWERTUNGEN (www.bgchemie.de)

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study investigated the developmental toxicity in Fischer 344 inbred rats of 2-ethylhexanoic acid (2-EHA) at the dose levels of 100, 250, and 500 mg/kg/day given by gavage. The treatment period with the test substance lasted from the gestational day 6 to the gestational day 15. The rats were sacrificed on gestational day 21. 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 abnormalities, head sections and skeletal evaluations

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexanoic acid

EC Number:

205-743-6

EC Name:

2-ethylhexanoic acid

Cas Number:

149-57-5

IUPAC Name:

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

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Virgin Fischer inbred albino [CDF (F344/crl/Br]
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY)
- Weight: males: 175 - 200 g; females: 130 - 150 g
- Housing: animals were housed individually or paired with the same sex in stainless-steel-wire-mesh cages.
- Diet (ad libitum): Prolab Certified Ground Rodent Chow, Agway, Inc., St. Marys, OH
- Water (ad libitum): water (Municipal Authority of Westmoreland County, Greensburg, PA)
- Quarantine period: 2 weeks

Selected animals were subjected to quality control screening (faecal sampling, histologic examination of selected organs, and serum viral antibody examination) prior to assignment to the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 - 22.2 °C
- Relative humidity: 45 - 65%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
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:

yes

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 analysed 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: male rats, paired with one female for mating, were used only once; the day of plug formation was designated gestational day (GD) 0 (Hafez, 1970)*
- Proof of pregnancy: vaginal plug

*Reference:
- Hafez, E.S.E. (1970). Reproduction and Breeding Techniques for Laboratory Animals Lea and Febiger, Philadelphia.

Duration of treatment / exposure:

Gestational day 6 - 15

Frequency of treatment:

daily

Duration of test:

21 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 250, and 500 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

25 pregnant rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Animal assignment: pregnant rats weighing 147 - 174 g 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 rats were orally dosed on gestational days 6 - 15 with 125, 250, 500, or 1000 mg 2-EHA/kg/day in corn oil in the range-finding study.
Results range-finding study:

1000 mg 2-EHA/kg/day:
- significant maternal toxicity was observed.
- seven of eight dams (87.5%) died between gestational day 7 and 9.
- clinical signs observed in this group during treatment consisted of ataxia, urogenital wetness, audible respiration, and red periocular encrustation.
- the one surviving dam had a fully resorbed litter.

500 mg 2-EHA/kg/day:
- the total level of postimplantation loss (early and late resorptions and dead foetuses) was increased and the corresponding percentage of live foetuses was decreased at 500 mg/kg/day.
- foetal body weights (both male and female) were reduced at 500 mg/kg/day but there were no external malformations or variations.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all females were examined once daily
- Cage side observations checked: clinical signs of toxicity, morbidity, and mortality

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: gestational days 0, 6 (for dose calculation), 12, 15, 18, and 21

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- 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 for the intervals gestational day 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, and 18 - 21.

WATER CONSUMPTION AND COMPOUND INTAKE: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 by CO2 inhalation
- Organs examined: the reproductive organs and abdominal and thoracic cavities were examined grossly.
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
- External examinations: Yes (viable foetuses)
- Soft tissue examinations: Yes, one-half of the foetuses from each litter were inspected for visceral abnormalities by modification of methods described by Staples (1974)*.
- Head examinations: Yes, the heads of foetuses used for visceral examinations 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, the remaining intact 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.

Statistics:

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.

Indices:

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 differences were observed between the three dose groups and controls for body weight, weight gain, and food consumption.
- clinical signs of toxicity, including ocular discharge and periocular encrustation, were increased in the 500 mg/kg/day dose group only.
- neither treatment-related necropsy findings in the dams on gestational day 21 nor differences in corrected terminal (minus gravid uterine weight) maternal body weight or gestational weight change (gestational day 21 - gestational day 0)
- liver weight (absolute and relative to corrected body weight) was increased at the high dose level (maternal liver weight: p < 0.01; relative maternal liver weight: p < 0.001).

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

250 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:yes

Details on embryotoxic / teratogenic effects:
- no difference between treated and control groups in the overall incidence of postimplantation loss (resorptions and dead foetuses) or percentage of viable foetuses.
- no difference in the incidence of external, visceral, or skeletal malformations between treated and control groups
- sex ratios were equivalent.

500 mg 2-EHA/kg/day:
- foetal body weights (both sexes) were reduced (p<0.001).
- growth retardation was also evidenced by a reduction in ossification of the axial and appendicular skeletons.
- an increase in the number of litters with foetuses exhibiting reduced ossification of cervical centra 5 and 6, anterior arch of the atlas, thoracic centra 1, 2, 3, 4, 12, and 13, extra 14th thoracic centrum, caudal segments, proximal phalanges of the forelimb and hindlimb, hindlimb metatarsals, and sternebrae 2, 5, and 6.
- dilation of lateral ventricles of the brain with tissue compression (a visceral malformation) was observed in six foetuses compared to two foetuses (in two litters) in the control group (not statistically significant).
- two variations were significantly increased in litters: dilation of the lateral ventricles of the brain with no tissue compression and extra (14th) thoracic centrum and arches.

250 mg 2-EHA/kg/day:
- an increase in the number of litters with foetuses with reduced ossification of the anterior arch of the atlas and proximal phalanges of the forelimb and hindlimb.
- dilation of lateral ventricles of the brain with tissue compression (a visceral malformation) was observed in one foetus compared to two foetuses (in two litters) in the control group (not statistically significant).

100 mg 2-EHA/kg/day:
- dilation of lateral ventricles of the brain with tissue compression (a visceral malformation) was observed in one foetus compared to two foetuses (in two litters) in the control group (not statistically significant).

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

According to the authors, the study revealed no teratogenic effects in Fischer 344 inbred rats following exposure to doses of 2-EHA during organogenesis. The no observed adverse effect level NOAEL) for developmental toxicity in rats is 100 mg/kg/day, based on slight foetotoxicity (reduced skeletal ossification) at 250 mg/kg/day. The corresponding maternal NOAEL is 250 mg/kg/day based on clinical sings of toxicity and increased liver weights at 500 mg/kg/day