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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
One treatment; animals followed for 144 hours
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable without restriction; acceptable and well-documented report which meets good scientific principles and was conducted according to GLPs.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The metabolic fate and disposition of 14^C di (2-ethylhexyl) terephthalate was investigated in male Sprague Dawley rats. The radioactive (14^C-labeled) test substance was mixed with unlabeled test substance in corn oil and was administered by gavage at a dose of 100 mg/kg bw. Rats were placed in glass metabolism chambers, and urine, feces, and expired air were collected periodically for up to 144 hours, after which time the rats were euthanized. Selected tissues were collected, and the tissues and carcasses were retained for radioactivity measurement. Total radioactivity was measured in excreta and tissues, and feces and urine were treated to characterize the metabolites of the test chemical.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Unlabeled test material:
-Test substance: Di (2-ethylhexyl) terephthalate
-Source: Tennessee Eastman Company (Kingsport, TN)
-Purity: 98% (by gas chromatography)

Radioactive test material:
-Test substance: [Hexyl-2-14^C] Di (2-ethylhexyl) terephthalate
-Source: New England Nuclear (Boston, MA)
-Purity: 97.7%
-Specific activity: 8.39 mCi/mmole
-Stability: Samples were analyzed by HPLC during the study and found to be chemically stable.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
other: CD(SD) COBS
Sex:
male
Details on test animals and environmental conditions:
Test animals:
-Source: Charles River Breeding Laboratories (Wilmington, MA)
-Sex: Male
-Acclimation period: 3-5 days
-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 glass metabolism cages (equipped to separate and collect expired air, urine and feces).
-Diet: Purina rodent chow #5001. Feed was provided, ad libitum, except when animals were fasted for 16 hours prior to dose administration. After test substance administration, feed was provided ad libitum to all animals except two rats. These two animals were fasted for an additional four hours, at which time they were provided feed ad libitum for the remainder of the study.
-Method of animal identification: uniquely numbered metal ear tags

Environmental Conditions:
-Temperature: 21.1 ± 0.9 °C
-Humidity: 50 ± 20% relative humidity

Study Dates:
-In-Life Experimental Start Date: February 20, 1981
-In-Life Experimental Termination Date: February 26, 1981

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Radiolabeled di (2-ethylhexyl) terephthalate ([Hexyl-2-14^C]di (2-ethylhexyl) terephthalate; specific activity 8.39 mCi/mmole) was mixed with non-labeled di (2-ethylhexyl) terephthalate and dissolved in corn oil to achieve the desired specific radioactivity. Rats weighing 200 to 300 grams were fasted for approximately 16 hours prior to dosing. Each animal was dosed by oral gavage with 100 mg/kg bw di (2-ethylhexyl) terephthalate. Feed was provided to eight rats immediately after dosing and withheld from the remaining two rats for four hours after dosing. Each rat received between 8.9 - 22.5 microCi of radioactivity.
Duration and frequency of treatment / exposure:
Duration of Test: 144 hours following a single dose
Doses / concentrations
Remarks:
Doses / Concentrations:
100 mg/kg bw
Each rat received between 8.9 - 22.5 microCi of radioactivity.
No. of animals per sex per dose:
10 males per dose
Control animals:
no
Details on study design:
Radiolabeled di (2-ethylhexyl) terephthalate ([Hexyl-2-14^C]di (2-ethylhexyl) terephthalate (specific activity 8.39 mCi/mmole) was mixed with non-labeled di (2-ethylhexyl) terephthalate and dissolved in corn oil to provide the dosing mixture. Ten rats weighing 200 to 300 grams were fasted for approximately 16 hours and were then administered a single oral dose of 100 mg/kg bw. Feed was provided to 8 of the rats immediately after dosing and withheld from 2 rats for four hours after dosing. Dehumidified air was drawn through the metabolism chambers at a rate of 500 mL/minute.

Expired air, urine, and feces were collected for up to 144 hours. At study termination, the animals were euthanized by cervical dislocation and selected tissues (brain, heart, lungs, liver, spleen, kidneys, testes, small and large intestines, and abdominal fat) as well as the carcass were collected and stored at -20 °C for subsequent analysis. A Tri-Carb Model 2660 Liquid Scintillation Spectrometer (Packard Instrument Co., Downers Grove, IL) was used for radioactivity measurements. Solid samples were combusted and measured for radioactivity using a model 306 sample oxidizer (Packard Instrument Co., Downers Grove, IL).
Details on dosing and sampling:
Expired air (14^CO2) was collected in two glass washing bottles connected in series with each containing 200-300 mL of 2.5 M NaOH. Traps were changed at specific intervals and aliquots from each trap were removed and measured for radioactivity. Metabolism chambers were rinsed with distilled water at each urine and fecal collection point, and in addition, the chambers were washed with methanol at the end of the collection period.

Samples of all fluids including urine, air traps and cage washes were analyzed for radioactivity. Urinary metabolites were characterized qualitatively by electron impact and chemical ionization gas chromatography with mass spectroscopy. Diluted urine was analyzed directly for terephthalic acid by high performance liquid chromatography (HPLC). Urine was also acidified with HCl and extracted continuously for 48 hours with diethyl ether. The remaining aqueous phase was treated with ß-glucuronidase to liberate glucuronic acid conjugates. The hydrolyzate was extracted continuously for 48 hours with diethyl ether. Sulfate conjugates were hydrolyzed by treating the remaining aqueous phase with 3N HCl. The hydrolyzate was extracted continuously for 48 hours with diethyl ether. The hydrolyzates were reduced in volume and analyzed by HPLC.

Feces from five of the eight rats allowed access to feed immediately after dosing and the two rats fasted for 4 hours after dosing were homogenized in 0.1 N HCl. Acetone was added to make a final mixture of 60% acetone/ 40% water and the mixture was centrifuged. Supernatant was removed and the residues were extracted twice with acetone. Combined extracts were analyzed for radioactivity. Fecal residues were dried, pulverized, combusted and counted. Feces from three of eight rats allowed access to feed immediately after dosing were extracted 3 times in acetone. Combined extracts and residues were analyzed for radioactivity. Fecal residues were extracted further by 24-hour continuous soxhlet extraction in acetone, and the extracts were analyzed for radioactivity. Fecal extracts were also analyzed by HPLC to characterize metabolites.

Tissues were extracted twice with acetone and the combined extracts analyzed for radioactivity. Residues were dried, powdered, combusted and counted. Carcasses were ground in a blender with acetone. The carcass mixture was refluxed and filtered. The residue was refluxed with n-hexane for at least 8 hours and filtered. The acetone and n-hexane filtrates were analyzed for radioactivity. Solid residues were pulverized and portions assayed for radioactivity.

The absorption half-life was estimated from the initial slope of the time course for the rate of excretion of expired 14^CO2. An approximation of the maximum amount of radioactivity residing in tissues was determined from the time course of excretion of radioactivity in expired air, urine, and feces.
Statistics:
Student's t-Test (where appropriate). Values of p ≤ 0.05 were considered statistically significant.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Di (2-ethylhexyl) terephthalate was rapidly hydrolyzed to 2-ethylhexanol, mono-(2-ethylhexyl) terephthalate, and unlabeled terephthalic acid and these metabolites were absorbed from the gastrointestinal tract. More than 36% of the administered dose was not absorbed and was excreted unchanged in the feces. This may be due to limited solubility or the availability of di (2-ethylhexyl) terephthalate to hydrolytic enzymes.
Details on excretion:
The mean total recovery of 14^C was 93.0 ± 2.2%. Most of the radioactivity was eliminated in the feces (56.5 ±12.1%) as di (2-ethylhexyl) terephthalate (36.6%), mono-(2-ethylhexyl) terephthalate (2.5%) and polar metabolites; in the urine (31.9 ± 10.9%) as metabolic products of mono-(2-ethylhexyl) terephthalate and 2-ethylhexanol; and a small amount was eliminated as expired 14^CO2 (3.6 ± 0.9%).

The rates of excretion of radioactivity in urine and feces revealed that more than 95 and 99% of the total urinary and fecal radioactivity was excreted by 24 and 48 hours, respectively. The time course for expired 14^CO2 was complex, with 14^CO2 appearing in expired air 45 minutes after the dose was given. Rate of excretion increased rapidly and reached a peak at 1.3 hours. Two additional peaks were noted at 2.8, and 5.0 hours after dosing. Approximately 68% of the total radioactivity in expired air was eliminated after 4 hours and the remaining 32% was eliminated after 40 hours.

The excretion of radioactivity in the feces, urine and expired air from the two animals for which food was withheld for 4 hours after dosing was comparable to the levels seen for animals given food immediately after dosing. An average of only 1.4 ± 0.6% of the administered dose remained in the carcass. Liver and adipose tissue had concentrations of radioactivity that were four and three times that of the carcass value, respectively.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
About 63% of the administered dose was metabolized to 2-ethylhexanol, mono-(2-ethylhexyl) terephthalate, and terephthalic acid and these compounds were absorbed from the gastrointestinal tract while the remainder of the dose was excreted unchanged in the feces. Di (2-ethylhexyl) terephthalate was metabolized predominantly to terephthalic acid. Radioactivity was eliminated in the feces (56 ± 12.1% of the dose) as di (2-ethylhexyl) terephthalate (36.6% of the dose), mono-(2-ethylhexyl) terephthalate (2.5%) and polar metabolites such as the glucuronic acid conjugate of 2-ethyl-5-hydroxyhexanoic acid. Several other uncharacterized, radiolabeled metabolites were also detected that were more polar than the parent compound.

Radioactivity was excreted in urine (31.9 ± 10.9%) as metabolites of mono-(2-ethylhexyl) terephthalate and 2-ethylhexanol and was expired in air as 14^CO2 (3.6 ± 0.97%). Major metabolites identified in urine included unlabeled terephthalic acid (equivalent to 51% of the dose on a molar basis), oxidized metabolites of 2-ethylhexanol and mono-(2-ethylhexyl) terephthalate, and glucuronic and sulfuric acid conjugates. The presence of unlabeled terephthalic acid indicates that complete hydrolysis of the diester had taken place. Based on hydrolytic evidence, about 17% and 12% of the urinary radioactivity was present as glucuronic and sulfuric acid conjugates, respectively. As many as eight urinary metabolites were detected by HPLC analysis including terephthalic acid, mono-(2-ethyl-4-carboxypropyl) terephthalate, mono-(2-(carboxymethyl) hexyl) terephthalate, 2-ethylhexanol, 2-ethylhexanoic acid, 5-hydroxy-2-ethylhexanoic acid, 5-hydroxy-2-ethylhexanolactone, and 2-heptanone.

Any other information on results incl. tables

The kinetic data indicate that about 92% of the administered dose was eliminated within 48 hours indicating that significant amounts of di (2-ethylhexyl) terephthalate or its metabolites are not retained by the rat. By the time of sacrifice, less than 2% of the dose remained in the carcass. Very low levels of 14^C were found in the tissues and most had relative levels lower than the carcass values. The exceptions were the liver, a probable site of continued metabolism, and the abdominal fat. More than 75% of the 14^C from the abdominal fat was acetone extractable suggesting this material would be eliminated.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study results
In an in vivo metabolism study, rats were administered a single dose of 100 mg/kg bw [14^C] di (2-ethylhexyl) terephthalate. Urine, feces and expired air were collected for up to 144 hours. All excreta, as well as selected tissues and carcass, were analyzed for radioactivity. The results of the study indicate that the major excretory products of di (2-ethylhexyl) terephthalate are unlabeled terephthalic acid in the urine and [14^C] di (2-ethylhexyl) terephthalate in the feces, together accounting for 87.1% of the dose. Approximately 3.6% of the administered radioactivity was expired as 14^ CO2. The metabolism of di (2-ethylhexyl) terephthalate was substantially different from its isomer, di (2-ethylhexyl) phthalate, in that the major metabolite of di (2-ethylhexyl) terephthalate was terephthalic acid and not oxidized metabolites of mono-(2-ethylhexyl) terephthalate.
Executive summary:

In a metabolism study using rats, radiolabeled di (2-ethylhexyl) terephthalate ([Hexyl-2-14^C]di (2-ethylhexyl) terephthalate (specific activity 8.39 mCi/mmole) was mixed with non-labeled di (2-ethylhexyl) terephthalate and dissolved in corn oil. Ten male CD COBS rats received a single dose of the test substance by oral gavage at a dose level of 100 mg/kg bw resulting in a received dose of between 8.9 - 22.5 microCi of radioactivity. The mean total recovery of 14^C was 93.0 ± 2.2%. Most of the radioactivity was eliminated in the feces (56.5 ± 12.1%) and urine (31.9 ± 10.9%), with smaller amounts in expired air (3.6 ± 0.9%). Approximately 1.4 ± 0.6% of the dose remained in the carcass. Metabolite analysis indicated that the major excretory products of di (2-ethylhexyl) terephthalate are terephthalic acid (TPA) and di (2-ethylhexyl) terephthalate, together accounting for 87.1% of the dose. Only a small percentage of the administered dose was excreted as mono-(2-ethylhexyl) terephthalate or its oxidative metabolites. Under the conditions of the study, di (2-ethylhexyl) terephthalate has a low potential for bioaccumulation and presents a low toxicity hazard.