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basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP compliant, minor restrictions in design but otherwise adequate for assessment

Data source

Referenceopen allclose all

Reference Type:
Pharmacokinetic studies with [2-14C-hexyl]2-ethylhexanoic acid (14C-EHA) in the female Fischer 344 rat
English, J.C., Deisinger, P.J., Perry, L.G., Guest, D.
Bibliographic source:
Toxicologist, 9, 87
Reference Type:
TOXICOLOGICAL EVALUATION No. 275 2-ethylhexanoic acid 06/00
BG Chemie
Bibliographic source:

Materials and methods

Objective of study:
Test guideline
equivalent or similar to
other: USEPA TSCA Health Effects Testing Guideline (CFR 40 798.7100)
GLP compliance:

Test material

Details on test material:
- 2-Ethylhexanoic acid (EHA): purity 99.6% (GC-MS), supplier: Eastman Chemical Products, Inc.
- [2-14C-Hexyl] EHA: specific radioactivity: 25 mCi/mmole. 14C-EHA accounted for 97.6% of the radioactivity as deterimined by HPLC or 100% as determined by GC.

Test animals

Fischer 344
Details on test animals and environmental conditions:
- Source: Charles River
- Weight at study initiation: 115-150 g
- Individual metabolism cages: yes
- Diet: Agway Prolab certified rodent diet; RMH 3000 while in holding cages, RMH 3200 while in metabolism cages; ad libitum except for a 4 hr period immediately after dosing
- Water: ad libitum
- Acclimation period: 2-3 days

Administration / exposure

Route of administration:
other: oral: gavage (acute and 14d); dermal; intravenous
other: cornoil (gavage); unchanged (dermal); physiological saline (i.v.)
Details on exposure:
- Gavage: Radiolabeled dose solutions were prepared by mixing the 14C-EHA with unlabeled EHA in corn oil such that each dose level contained approximately 10 µCi. At 100 mg/kg, 5% EHA solutions in corn oil were prepared and rats were given approximately 2 ml/kg. At 1000 mg/kg, 20% EHA solutions in corn oil were prepared and rats were given approximately 5 ml/kg. In the repeated oral dose study, rats were treated for 14 days with unlabeled EHA and with 14C-EHA on the 15th day.
- Dermal: Dorsal surfaces of the rats were shaved and annular columnators for the dose solution were secured to the shaved surfaces with tissue adhesive. Rats were treated with undiluted 14C-EHA, applied with a hypodermic syringe into the columnator, covered with a plastic film. Each rat was given approximately 10 µCi. The plastic film was sealed, the animals were wrapped with bandaging tape and the application site remained occluded for 96 hr.
- Intravenous: 14C-EHA was mixed with unlabeled EHA in physiological saline (0.9% NaCl) such that each dose contained approximately 10 µCi in 0.5 ml. 1 mg/kg 14C-EHA was injected into the tail vein and pressure was applied to the injection site for 30 seconds. If there was evidence of distenstion surrounding the injection site due to subcutaneous fluid accumulation, the animal was not used in the study.
- Skin washing efficiency study: Rats were anesthetized, columnators were secured dorsally and undiluted 14C-EHA (1000 mg/kg; approximately 10 µCi) was applied to the skin. After 5 min, test material was removed by aspiration with a pipette, the application site was washed sequentially with 19 cotton swabs that had been saturated with 40% pHisoDerm (Winthrop) in warm water. The application site was rinsed 5 times with distilled water and dried. Animals were wrapped with bandaging tape and placed in glass metabolic chambers.
Duration and frequency of treatment / exposure:
Gavage: single dose or once daily for 15 days
Dermal: 96 hr (occluded)
Intravenous: single dose
Skin washing efficiency: 5 min

Doses / concentrations
Doses / Concentrations:
Gavage, acute: 100 and 1000 mg/kg
Gavage, repeated: 100 mg/kg
Dermal: 100 and 1000 mg/kg
Intravenous: 1 mg/kg
Skin washing efficiency: 1000 mg/kg
No. of animals per sex per dose:
Gavage, acute: 8
Gavage, repeated: 4
Dermal: 8
Intravenous: 8
Skin washing efficiency: 4
Control animals:
Positive control:
Details on dosing and sampling:
Urine was collected over dry ice at 8 and 24 hr and then at 24 hr intervals vor 96 hr. Urine volume was recorded and counted by LSS. Remaining urine was frozen to permit subsequent analysis by HPLC or GC-MS. Feces were collected at 8 and 24 hr and then at 24 hr intervals vor 96 hr. Samples were weighed and frozen until analysis for radioactivity (LSS counting). Orbital blood was collected of 4 animals of the low oral dose, low dermal dose, and intravenous dose groups at 0.25, 0.5, 1, 8, 24, 48 and 96 hr after dosing and analyzed by LSS. Metabolism chambers were rinsed at 24 hr intervals for 96 hr and radioactivity in the rinsings was assayed (LSS). At 96 hr after dosing, animals were killed (CO2) and body weights were recorded.

- 0.2 mg/ml ß-Glucuronidase was added (pH 5.5; acetate buffered), incubated for 16 hr at 37ºC, filtered and analyzed by HPLC.
- 45 mg/ml sulfatase was added (pH 5.5; acetate buffered), incubated for 16 hr at 37ºC, filtered and analyzed by HPLC.
- Hydrochloric acid - urine containing 1.5 N-HCl was incubated for 1 hr in a boiling water bath, neutralized, filtered and analyzed by HPLC.
In preparation for GC-MS analysis, selected urine samples or fractions collected from multiple HPLC runs were extracted at pH 3 with ethyl acetate. The extracts were concentrated and analyzed by GC-MS with or without prior derivatization by methylation or trimethylsilylation.

Pharmacokinetic parameters were derived for the total 14C label in blood after intravenous, 100 mg/kg oral, and 100 mg/kg dermal administration using a computer program for nonlinear regression analysis, NONLIN84. Data were modeled using curves that minimized the sums of squares of the residuals.

Skin washing efficiency study: to calculate the total 14C recovered by the washing procedure, the following components were analyzed for radioactivity by LSS: the test material recovered from the skin surface, pooled rinsings, the plastic tip used in recovering the test material, and the cotton swabs.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Blood levels after intravenous injection appear to decay in a triphasic manner with half‑lives of 0.19 ± 0.11 hrs, 6.6 ± 3.9 hrs, and 117 ± 47 hrs. After oral administration, peak blood levels were achieved after 15 or 30 minutes, and also declined triphasically with half‑lives similar to what had been estimated from intravenous administration (0.32 ± 0.04 hrs, 6.8 ± 3.5 hrs, and 98.2 ± 32.8 hrs). Dermal application resulted in slower absorption with peak blood levels occurring 5.7 ± 0.4 hours after application and a half‑life of 3.2 ± 0.1 hr. Elimination was biphasic with half-lives of 4.2 ± 0.2 and 251 ± 135 hrs.
Details on excretion:
Approximately 72-75% of the oral dose was excreted in the urine within 24 hours. Little radioactivity (<10%) was excreted after 24 hours. The dose influenced the rate of excretion such that 50% of the radioactivity was excreted in the first 8 hours after the 100 mg/kg dose versus 20% after the 1000 mg/kg dose. Fecal excretion accounted for 7-12% in both cases. Slightly less radioactivity was excreted as either urine (64%) or feces (2%) after intravenous injection. Repeated dosing with unlabeled 2-ethylhexanoic acid altered excretion of radioactivity to approximately 55% in urine and 15% in feces within the first 24 hours. After dermal application, approximately 30% of the dose was excreted in the urine during the first 24 hours followed by an additional 8 or 17% from 24-96 hours for the 100 and 1000 mg/kg doses, respectively. Fecal excretion was 7% regardless of the dose level. Dermal absorption was estimated to be 63-70% relative to intravenous administration.

Metabolite characterisation studies

Metabolites identified:
Details on metabolites:
Analysis of urine indicated three major peaks: one as a glucuronide conjugate of 2‑ethylhexanoic acid; one as a glucuronide conjugate of hydroxylated and diacid derivatives of 2-ethylhexanoic acid, possibly 2-ethyl-6-hydroxyhexanoic acid and 2‑ethyl-1,6-hexanedioic acid; and the last as unmetabolized 2-ethylhexanoic acid. No sulfate derivatives were detected. The percentages of each metabolite changed with the dose and route of administration (see Table in remarks on results section).

Any other information on results incl. tables

 Table - Overview of metabolite excretion



Percentage excreted as


1000 mg/kg

45% glucuronide-2-ethylhexanoic acid



7% glucuronide-diacid or hydroxylated 2-ethylhexanoic acid



2% unmetabolized 2-ethylhexanoic acid


100 mg/kg

20% glucuronide-2-ethylhexanoic acid



14% glucuronide-diacid or hydroxylated 2-ethylhexanoic acid



7% unmetabolized 2-ethylhexanoic acid


100 mg/kg

12% glucuronide-2-ethylhexanoic acid



12% glucuronide-diacid or hydroxylated 2-ethylhexanoic acid



5% unmetabolized 2-ethylhexanoic acid


1000 mg/kg

17% glucuronide-2-ethylhexanoic acid



3% glucuronide-diacid or hydroxylated 2-ethylhexanoic acid



3% unmetabolized 2-ethylhexanoic acid


100 mg/kg

4% glucuronide-2-ethylhexanoic acid



9% glucuronide-diacid or hydroxylated 2-ethylhexanoic acid



2% unmetabolized 2-ethylhexanoic acid

Applicant's summary and conclusion