Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Hydrolysis, absorption and metabolism of di (2-ethylhexyl) terephthalate in the rat

Author:

Barber E. D. et al.

Year:

1994

Bibliographic source:

Xenobiotica 24 (5), 441-450

Materials and methods

Objective of study:

other: hydrolysis

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
In this study the rate of hydrolysis of di(2-ethylhexyl)terephthalate (DEHT) by rat intestinal enzymes was evaluated.

- Short description of test conditions:
The hydrolysis of di(2-ethylhexyl) terephthalate (DEHT) and di(2-ethylhexyl) phthalate (DEHP) were studied using rat gut homogenate fractions in vitro.
The test substances were first dissolved in methanol and was then added to the intestinal homogenate. The mixture was incubated at 37 °C for up to 30 minutes. Samples were removed periodically and mixed with 1 mL of 6 N HCl to inactivate the intestinal enzymes. The samples were extracted continuously for 24 hours with diethyl ether and were then concentrated and analysed by capillary gas chromatography.

- Parameters analysed / observed:
The half-life for disappearance was measured

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) terephthalate

EC Number:

229-176-9

EC Name:

Bis(2-ethylhexyl) terephthalate

Cas Number:

6422-86-2

Molecular formula:

C24H38O4

IUPAC Name:

bis(2-ethylhexyl) terephthalate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Eastman Chemical Co.
- Purity: > 98%

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Wilmington, MA, USA)
- Weight at study initiation: 200 – 300 g
- Housing: Single housed in suspended wire-mesh cages prior to dosing. After test substance administration the rats were housed in Jencons (Hemel Hempstead, UK) glass metabolism cages equipped for the efficient separation of expired air, urine and faeces.
- Diet: Ralston Purina rodent chow #5001. Feed was provided, ad libitum, except when animals were fasted for 16 hours prior to dose administration.
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 ± 0.9
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

other: in vitro use of rat gut homogenate fractions

Vehicle:

other: methanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
DEHT stock solutions were prepared in methanol and added to 60 ml intestinal homogenate maintained at 37 °C in a water bath. Methanol was added to an aliquot of intestinal homogenate as a negative control. Samples (10 ml) were withdrawn at 0, 5, 10, 20 and 30 min and immediately treated with 1 mL 6xNHCI and 10 mL water. Unchanged DEHT and its metabolites were partitioned into diethyl ether by 24-h continuous extraction. Extracts were then reduced in volume, dried with MgSO4, and diluted with ethylacetate or methanol. Samples were analysed for 14C using a Packard Tri-Carb" liquid scintillation spectrometer and Kodak Ready-to-Use II scintillant.

Duration and frequency of treatment / exposure:

single application

Doses / concentrations

Remarks:

not specified

No. of animals per sex per dose / concentration:

n.a

Control animals:

yes, concurrent vehicle

Details on study design:

Gut homogenates were prepared from fasted (16 h) male Sprague-Dawley rats weighing 200-300 g. Following killing, the small intestines were removed, placed in 3 vol of ice-cold 0.2 M phosphate buffer, pH 7.0, and homogenized for 2-3 min at 6000 rpm using a Brinkman Polytron' homogenizer. 'I'he homogenate was centrifuged at 1027 g for 20 min in a Sorvall RC-3R refrigerated centrifuge. DEHT stock solutions was prepared in methanol and added to 60 ml intestinal homogenate maintained at 37 °C in a water bath. Methanol was added to an aliquot of intestinal homogenate as a negative control.
Samples (10 ml) were withdrawn at 0, 5, 10, 20 and 30 min and immediately treated with 1 m1 6 NHCI and 10 ml water. Unchanged DEHT and their metabolites were partitioned into diethyl ether by 24-h continuous extraction. Extracts were reduced in volume, dried with MgSO4,, and diluted with ethyl acetate or methanol. Samples were analysed for 14C ' using a Packard Tri-Carb" liquid scintillation spectrometer and Kodak Ready-to-Use II scintillant.
Homogenate extracts were analysed by tlc on silica gel 60 F-254 plates developed in chloroform -methanol-acetic acid (143 : 7: 2). The developed plates were divided into eight equal zones, and the silica gel from each zone mas quantitatively removed from the plate and eluted with methanol. Samples of the methanol extracts were counted by liquid scintillation spectrometry as described above. Aliquots (if the tlc plate extracts were chromatographed using a Waters hplc system equipped with a 4.6 mm x 25 cm Zorbax" ODS column. Chromatography was performed using water and methanol, each containing 0.09% formic acid. A variety of gradients were used to separate the esters and their metabolites. Hplc column eluents were collected using a Multirac fraction collector and were counted by liquid scintillation spectrometry.
The diesters and their metabolites in the homogenate extracts were also analysed by gc using a Hewlett-Packard gc equipped with a flame ionization detector and a bonded-phase DB-1 column.

Results and discussion

Main ADME results

Main ADME results 1

Type:

metabolism

Results:

1.97 mol of 2-ethylhexanol (2-EH) produced from 1 mol DEHT after 30 minutes

Main ADME results 2

Type:

metabolism

Results:

half-life for disapperance of DEHT homogenates of rat small intestines is 53.3 minutes

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The production of 2-ethylhexanol (2-EH) increased with time over the 30-minute incubation period. The stoichiometry of the hydrolysis reactions indicated that di(2-ethylhexyl)terephthalate (DEHT) was converted to 2-EH and showed that at 30 min 1.97 mols of 2-EH are produced from 1 mol DEHT. The half-life for the disappearance of DEHT was estimated to be 53.3 min.

Any other information on results incl. tables

Table 1: Production of 2-ethylhexanol and the disappearance of DEHT from rat small intestine homogenate preparations.

Results are expressed as µmol/kg homogenate

Time (min)	2-EH produced	DEHT consumed
0	0	0
5	11.3	23.7
10	33	16.4
20	33	32.4
30	51.5	26.2

Applicant's summary and conclusion

Conclusions:

In an in vitro study, DEHT was incubated with rat intestinal homogenate. At specific intervals, samples were removed and analyzed for parent compound and metabolites. Under the study conditions, it was determined that DEHT was metabolized by enzymes present in the gut to 2-ethylhexanol and that the hydrolysis followed a first-order kinetics with a disappearance half-life of 53.3 minutes. The calculated stoichiometry at 30 min showed that 1.97 mol ethylhexanol is formed per mol DEHT.

Executive summary:

In an in vitro study to determine the rate of hydrolysis of di(2-ethylhexyl)terephthalate (DEHT) by enzymes in the gut,the test substance was incubated in vitro with rat intestinal homogenates. The test substance was dissolved in methanol and was then incubated at 37 °C for up to 30 minutes with these gut homogenates. Periodically samples were drawn, and the intestinal enzymes were inactivated. Under the study conditions, it was determined that DEHT was metabolized by enzymes present in the gut to 2-EH and that the hydrolysis followed a first-order kinetics with a disappearance half-life of 53.3 minutes. The calculated stoichiometry at 30 min showed that 1.97 mol ethylhexanol is formed per mol DEHT.