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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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented GLP study which meets basic scientific principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1998

Materials and methods

Objective of study:
other: to determine the rate of hydrolysis of ethyl acetate in male rats in vivo and in vitro
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The hydrolysis of ethyl acetate was monitored by following the decline in [14C]ethyl acetate and increase in [14C]ethanol and [14C]acetic acid concentrations in blood following an intravenous (iv) dose. Similarly for the brain kinetic studies, concentrations of [14C]ethyl acetate, [14C]ethanol, and [14C]acetic acid in brain tissue were also determined. In addition, the in vitro hydrolysis rate of ethyl acetate in whole blood was determined by measuring the decline in [14C]ethyl acetate concentrations in blood spiked with micromolar concentrations of [14C]ethyl acetate. Together, these studies provide kinetic information on the in vivo systemic hydrolysis and in vitro blood hydrolysis of ethyl acetate in the rat.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): [14C] ethyl acetate
- Radiochemical purity (if radiolabelling): 99.73% (Vendor Assay)
- Specific activity (if radiolabelling): 3.10 mCi/mmol
- Locations of the label (if radiolabelling): 1-ethyl[14C]
- Storage condition of test material:
- Other: The [14C]ethyl acetate was diluted to a specific radioactivity of 0.42 mCi/mmol with unlabeled ethyl acetate. The mass purity of the diluted sample was determined to be > 99%.
Radiolabelling:
yes
Remarks:
1-ethyl[14C] acetate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Zivic-Miller Laboratories, Inc., Zelienople, PA
- Age at study initiation: young adult
- Weight at study initiation: 266 and 402 g
- Housing: Prior to the studies, animals were housed in wire-mesh, stainless-steel cages.
- Individual metabolism cages: yes/no
- Diet (e.g. ad libitum): certified rodent diet (PMI, Inc. Rodent 5002 Pellet) ad libitum
- Water (e.g. ad libitum): domestic tap water ad libitum
- Other: The animals were surgically prepared by the vendor with either femoral and jugular vein cannulae (probe and blood kinetic studies), or femoral vein cannulae only (brain kinetic studies). Additional animals were obtained without surgical alteration (in vitro blood kinetic studies).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 deg
- Humidity (%): 40 to 67%
- Air changes (per hr): at least 10 to 15
- Photoperiod (hrs dark / hrs light): room lighting following a 12-hour light/dark cycle.

Administration / exposure

Route of administration:
other: intravenous and in vitro
Vehicle:
other: saline in vivo
Details on exposure:
Exposures were to a saline solution of [14C]ethyl acetate.
Duration and frequency of treatment / exposure:
Intravenous studies: single bolus dose

In vitro studies: incubation for 120 minutes
Doses / concentrations
Remarks:
Doses / Concentrations:
Intravenous Blood Kinetic Studies: 100 or 10 mg/kg (3.75 ml/kg)
Intravenous Brain Kinetic Study: 100 mg/kg (3.75 ml/kg)
In vitro Blood Study: the highest concentration (400 ug/g) seen in blood following the high dose (100 mg/kg) iv administration in vivo
No. of animals per sex per dose:
Intravenous Blood Kinetic Studies: 5 male rats per dose level
Intravenous Brain Kinetic Study: 4 male rats per sample interval
In vitro Blood Study: blood from 4 untreated male rats
Control animals:
no
Positive control:
No
Details on study design:
Initial studies determined that due to respiratory and circulatory depression caused by larger bolus intravenous doses of ethyl acetate in saline, the high dose level for the blood and brain kinetic studies would be 100 mg/kg.
Details on dosing and sampling:
Intravenous Blood Kinetic Studies: Groups of five male SD rats were administered a saline solution of [14C]ethyl acetate as a bolus via the femoral vein cannula at 100 or 10 mg/kg dose levels (3.75 ml/kg). The time of dose was recorded as the time the ethyl acetate administration was completed. Serial blood samples were collected with a heparinized syringe (5 IU/ml sodium heparin) from the jugular vein cannula of each animal at 8 time points ranging from 30 to 540 sec following the dose administration. Following deproteinization, blood concentrations of [14C]ethyl acetate, [14C]ethanol, [14C]acetaldehyde, and [14C]acetic acid were determined by HPLC/Rad. Total [14C] concentrations in whole blood and in deproteinized blood were
determined by liquid scintillation counting (LSC, LKB 1217, LKB Instruments, Inc., Gaithersberg, MD).

Intravenous Brain Kinetic Study: Groups of four male SD rats were administered a saline solution of [14C]ethyl acetate as a bolus via the femoral vein cannula at 100 mg/kg (3.75 ml/kg). The time of dose was recorded as the time the ethyl acetate administration was completed. The animals were
euthanatized by exsanguination under C02 anesthesia at each of four time points from approximately 30 to 300 s following dose administration. The brain was excised, homogenized in ice cold saline, and deproteinized. The C02 anesthesia time and the brain homogenate deproteinization time was accurately recorded. Concentrations of [14C]ethyl acetate, [14C]ethanol, [14C]acetaldehyde, and [14C]acetic were determined in the deproteinized brain homogenates and in deproteinized whole blood by HPLC/Rad. Total [14C] concentrations in whole blood and brain homogenates and in deproteinized blood and brain homogenates were determined by LSC.

In vitro Blood Study: Whole blood was collected from the vena cava of four untreated male SD rats (not surgically altered) following C02 anesthesia. Blood coagulation was inhibited by the addition of sodium heparin. The blood samples were spiked with [14C]ethyl acetate in saline to give a final ethyl acetate concentration approximating the highest concentration (400 ug/g) seen in blood following the high dose (100 mg/kg) iv administration in vivo. The blood samples were placed in a 37°C shaking incubator and sampled periodically from 2 to 120 min following the [14C]spike. Following deproteinization, concentrations of [14C]ethyl acetate, [14C]ethanol, and [14C]acetic were determined by HPLC/Rad. Total [14C] concentrations in whole blood and deproteinized blood were assayed by LSC.
Statistics:
A nonlinear least squares data-fitting program (PKAnalyst®, Version 1.0, MicroMath Scientific Software, Salt Lake City, Utah, 1995) was used to derive pharmacokinetic parameters for blood and brain ethyl acetate concentrations.

For all of the studies, a monoexponential equation of the form

Ct = Dose/VD X e -Kelim t

was fitted to individual values of concentration-time data, where Ct is the concentration in blood at time t, VD is the apparent volume of distribution, and Kelim is the elimination rate
constant. The elimination half-life of ethyl acetate was calculated as follows:

t1/2 =1n2 / Kelim

Results and discussion

Preliminary studies:
Initial studies determined that due to respiratory and circulatory depression caused by larger bolus intravenous doses of ethyl acetate in saline, the high dose level for the blood and brain kinetic studies would be 100 mg/kg.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
In vivo exposures were by intravenous injection.
Details on distribution in tissues:
Not studied
Details on excretion:
Not studied
Toxicokinetic parameters
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: Elimination half-lives of 33.4 s and 36.9 s were estimated for the 10 and 100 mg/kg doses, respectively.

Metabolite characterisation studies

Metabolites identified:
no

Any other information on results incl. tables

Following both the high and low dose bolus iv injections, a rapid distribution and equilibration phase was followed by very rapid elimination of the parent compound.  Elimination half-lives of 33.4 s and 36.9 s were estimated from the first order elimination rate constants of 0.0208/s and 0.0188/s for the 10 and 100 mg/kg doses, respectively.  Evidence that the carboxyesterase capacity was not saturated at the high dose level is found from the similar elimination rates for these two dose levels.  Since depression of central nervous system function is noted following inhalation of ethyl acetate, concentrations of ethyl acetate and metabolites in brain tissue homogenates were assayed following the 100 mg/kg intravenous dose.  Total [14C] concentrations in brain homogenates were approximately 75% of those seen in the blood following the 100 mg/kg administrations, suggesting that ethyl acetate and metabolites are not preferentially sequestered in this tissue.  In addition, ethyl acetate in the brain was rapidly hydrolyzed (kelim = 0.0285/s), and the ethanol formed was rapidly eliminated evidence that supports the use of ethanol data in ethyl acetate risk assessment.  An in vitro ethyl acetate blood kinetic study, conducted at approximately the same concentration as measured in the initial 100 mg/kg in vivo study samples, yielded an estimated elimination rate constant of 0.0298/min, only a fraction of that estimated from the in vivo studies.  This indicates that systemic organ carboxyesterase activity is predominant in the in vivo hydrolysis of ethyl acetate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: ethyl acetate is rapidly hydrolyzed in vivo
Executive summary:

The rate of hydrolysis of ethyl acetate in male rats in vivo and in vitro was studied by Deisinger and English (unpublished). Ethyl acetate was rapidly hydrolyzed to ethanol following intravenous injection in rats, with an in vivo elimination half-life in blood of 33-37 seconds.

Carboxyesterase capacity was not saturated at 100 mg/kg. There was no evidence that ethyl acetate or metabolites were preferentially sequestered in brain, based on total [14C] levels. The rapid in vivo hydrolysis of ethyl acetate to ethanol supports the use of ethanol systemic toxicity data in evaluating the potential effects of ethyl acetate exposure.