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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not conducted according to guideline/s and GLP but the report contains sufficient data for interpretation of study results

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
In vitro hydrolysis in liver homogenates of the two primary constituents of primary amyl acetate was examined.
GLP compliance:
no
Remarks:
While the procedures and techniques used during the conduct of this study generally meet GLP standards, no part of the study has been audited or approved by the Quality Assurance Unit at Bushy Run Research Center.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Five mCi of [l-l4C]-n-pentyl acetate (specific activity = 6.9 mCi/mmol), Lot No. 900301, CAS No. 6628-63-7, was received on March 2, 1990 from Wizard Laboratories (Davis, CA), and assigned BRRC Sample No. 53-84. The substance was a clear liquid. The substance was stored frozen at -20°C. The purity of the substance was stated to be 99% by the supplier.

Five mCi of [1-14C]-2-methylbutyl acetate (specific activity = 6.9 mCi/mmol), Lot No. 900302, CAS No. 624-41-9, was received on March 5, 1990 from Wizard Laboratories (Davis, CA), and assigned BRRC Sample No. 53-93. The substance was a clear liquid. The substance was stored frozen at -20°C. The purity of the substance was stated to be 98% by the supplier.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Because of the experimental nature of the preliminary testing experiments, animals were ordered as needed for use in the study. Animals ordered for the study which were not used within the age group specified for the study (10-15 weeks) were sacrificed after 15 weeks of age. The first group of animals, consisting of 12 male rats, arrived on October 22, 1990 from Harlan Sprague Dawley Inc. (Indianapolis, IN). They were designated by the supplier to be approximately 9 weeks old (the birth date was recorded as August 20, 1990) upon arrival. The second group of animals, consisting of 6 male rats, arrived on December 29, 1991 from Harlan Sprague Dawley Inc. (Indianapolis, IN). They were designated by the supplier to be approximately 9 weeks old (the birth date was recorded as October 28, 1991) upon arrival. The third group of animals, consisting of 12 male rats, arrived on January 27, 1992 from Harlan Sprague Dawley Inc. (Indianapolis, IN). They were designated by the supplier to be approximately 7 weeks old (the birth date was recorded as December 9, 1991) upon arrival. The fourth group of animals, consisting of 60 male rats, arrived on March 24, 1992 from Harlan Sprague Dawley Inc. (Indianapolis, IN). They were designated by the supplier to be approximately 6 weeks old (the birth date was recorded as February 2, 1992) upon arrival. The fifth group of animals, consisting of 8 male rats, arrived on July 28, 1992 from Harlan Sprague Dawley Inc. (Indianapolis, IN). They were designated by the supplier to be approximately 9-10 weeks old (the birth date was recorded as May 26,
1992) upon arrival. The sixth group of animals, consisting of 6 male rats, arrived on September 8, 1992 from Harlan Sprague Dawley Inc. (Indianapolis, IN). They were designated by the supplier to be approximately 9 weeks old (the birth date was recorded as July 7, 1992) upon arrival. The seventh group of animals, consisting of 6 male rats, arrived on April 6, 1993 from Harlan Sprague Dawley Inc. (Indianapolis, IN). They were designated by the supplier to be approximately 9 weeks old (the birth date was recorded as February 2, 1993) upon arrival. Two of the rats from this last shipment were used for this study, and the remainder were used for other studies.

The acclimation period was 3 days. Within 2 days of receipt, the animals were examined by a Clinical Veterinarian. Based on the results of the examination, the Clinical Veterinarian indicated that these animals were in good health and suitable for use. All animals were assigned unique numbers and identified by ear tags. Prior to use in the study, animals were housed in stainless steel cages with wire mesh floors. DACB* (Deotized Animal Cage Board; Shepherd Specialty Papers, Inc.) was placed under each cage and changed at least 3 times per week.

An automatic timer was set to provide fluorescent lighting for a 12-hour photoperiod (approximately 0500 to 1700 hours for the light phase).
Temperature and relative humidity were recorded (Cole-Parmer Hygrothermograph Seven-Day Continuous Recorder, Model No. 8368-00, Cole-Parmer Instrument Co., Chicago, IL). Temperature was routinely maintained at 65-77OF; relative humidity was routinely maintained at 40-70%.

Tap water (Municipal Authority of Westmoreland County, Greensburg, PA) was available ad libitum and was delivered by an automatic watering system with demand control valves mounted on each rack. Water analyses were provided by the supplier, Halliburton NUS Environmrntal Laboratories, Materials Engineering and Testing Co., and Lancaster Laboratories, Inc., at regular intervals. EPA standards for maximum levels of contaminants were not exceeded. Pelleted, certified AGWAY PROLAB Animal Diet Rat, Mouse, Hamster 3000 (Agway Inc.) was available ad libitum.

Administration / exposure

Route of administration:
other: in vitro
Vehicle:
DMSO
Details on exposure:
Rat liver homogenates were prepared fresh on the day of each experiment in the following manner: 50 mM sodium phosphate buffer solution (pH 7.4) was prepared by adding approximately 6.9 g of (monobasic) sodium phosphate (MW=137.99) to a 1-liter volumetric flask and diluting to volume with Milli-Q water. The solution was transferred to a 1000 ml beaker, a stir bar was added, and the pH adjusted to 7.4 (while stirring on a magnetic stir plate) by drop-wise addition of concentrated hydrochloric acid or sodium hydroxide. For the liver homogenate preparation, a Fischer 344 male rat was sacrificed by CO2 asphyxiation, the liver removed, and the carcass discarded. A 20% (w/v) homogenate was prepared by first weighing approximately 10 g of excised liver, which was then cut, minced, and rinsed with 50 mM sodium phosphate buffer (pH 7.4). Minced liver was then added to approximately 40 ml of 50 mM sodium phosphate buffer (pH 7.4) in a 50 ml glass homogenizing tube. The liver was homogenized using a Polytrono Homogenizer for approximately 15 seconds at a speed setting of 4, then placed on ice until use. Dilutions (v/v) of the 20% liver homogenate were made as needed.

Preparation of Stock and Spiking Solutions
Upon receipt, stock solutions of 14C-n-pentyl acetate and 14C-2-methylbutyl acetate were prepared by diluting each with approximately 250 yl of dimethyl sulfoxide (DMSO), and the weights of both the test substances and DMSO added to each were recorded. For some experiments, the stock solutions were used to spike the incubations with the appropriate concentrations. For experiments 61 requiring lower substrate concentrations, spiking solutions were prepared by diluting the appropriate amount of radiolabeled isomer (g) with the appropriate amount of DMSO (g). The concentrations of the incubations were calculated using the known specific activity of the radioactive test substances and the actual weights of stock solution and DMSO added.

Preliminary Testing
For the following experiments, the liver homogenate and stock or spiking solutions were prepared as described above.

Experiment 1 consisted of timed incubations of 25 mM n-pentyl acetate and 2-methylbutyl acetate in 20% liver homogenate, inactivated 20% liver hornogenate, and buffer. The liver homogenate was prepared as described above and a portion was heat-inactivated at 70°C for 1/2 hr. Approximately 22 ul of each stock solution was added to 5 ml HPLC vials capped with a foil-lined Teflon cap, to which 2 ml of either 20% (active or inactive) liver homogenate or buffer were added to achieve concentrations of approximately 25 mM. The samples were then incubated in a 37C water bath with continuous agitation, and each was sampled at 0, 5, 15, 30, 45, 60, 75, 90, 105, and 120 minutes. Fifty ul of liver homogenate or buffer and 10 ul of trichloroacetic acid (TCA) (100%, w/v) were transferred by pipette into a 400 ul microcentrifuge tube. The tube was mixed briefly, then centrifuged in an Eppindorf centrifuge at 14,600 rpm for 5 minutes. The supernatent was transferred to an HPLC vial, equipped with a 300 ul polyspring insert, and 50 u1 were injected onto the HPLC system described in the analytical chemistry section. The identity of the acetates and alcohols was confirmed by comparison with authentic 14C-radiolabeled standards, which were analyzed concurrently with the samples.

For Experiment 2, the effects of TCA or acetonitrile on the hydrolysis 2-methylbutyl acetate incubated with 20% liver homogenate were determined. For the experiment, 1 vial containing 2 ml of 20% liver homogenate and 2 vials each containing 2 ml of buffer were spiked to achieve concentrations of 2 mM by adding 2 ul of the 2-methylbutyl acetate stock solution. The spiked liver homogenate and one of the spiked buffer solutions were incubated in a 37C water bath with continuous agitation. The remaining spiked buffer solution was kept at room temperature (approximately 25C) for the duration of the experiment. Samples were withdrawn in duplicate from each of the vials at 5, 15, 30, 45, and 60 minutes, and transferred into polyethylene microcentrifuge tubes containing 100 ul of either TCA or acetonitrile. The samples were processed as described for Experiment 1, and 10 ul were injected onto the HPLC system.

In Experiment 3, steps were taken to minimize losses due to evaporation of the test substances or products by reducing the volumes used, performing the incubations in small septum-sealed containers, and spiking the incubations and transferring the supernatents only by syringe. In addition, the effect of pH on the autohydrolysis of 2-methylbutyl acetate in phosphate buffer was determined. For the experiment, 3 vials each containing 2 ml of buffer at pH 6.0, 8.1, and 7.4., 1 vial containing 2 ml of water, and 1 vial containing 0.9% saline were spiked to achieve concentrations of 2 mM by adding 2 ul of the 2-methylbutyl acetate stock solution. The spiked solutions were then incubated in a 37C water bath with continuous agitation for 10 minutes, an aliquot was transferred to an HPLC vial, and 10 ul were injected onto the HPLC.

Experiment 4 was conducted to determine that the hydrolysis noted in previous experiments was enzymatic. n-Pentyl acetate (25 mM) was incubated with either 20% liver homogenate or buffer. Duplicate aliquots were withdrawn at intervals of 5, 15, 30, 45, and 60 minutes, and added to polyethylene vials containing either methanol or TCA (for comparison). The samples were processed as described for Experiment 1, and 10 ul were injected onto the HPLC system.

Experiments 5-9 were conducted to determine the proper agent for the inhibition of enzyme activity.

Experiment 5 was conducted by using 2 ul of either n-pentyl acetate or 2-methylbutyl acetate (25 mM) which were added to vials, each containing 2 ml of either 20% liver homogenate, heat-inactivated liver homogenate, or buffer. For this experiment, glass beads were added to the incubation mixture to aid in mixing. The samples were incubated in a shaking water bath at approximately 37C. Aliquots were withdrawn from the incubations at intervals of 5, 15, 30, 45, and 60 minutes, and added to polyethylene vials containing methanol. The samples were processed as described for Experiment 1, and 10 ul were injected onto the HPLC system.

Experiment 6 was essentially a repeat of Experiment 5, with the exception that the liver homogenate was inactivated by heating at 70°C for 1 hour instead of 30 minutes.

Experiment 7 was performed to reproduce the results of Experiment 6, and was identical in design to Experiment 6, but the results were inconclusive, as a problem with the HPLC system prevented analysis of the samples.

Experiment 8 was identical in design to Experiment 6, with the exception that sodium dodecyl sulfate (SDS) was used instead of heat-treatment to inactivate the liver homogenate.

Experiment 9 had essentially the same experimental design as the previous three experiments, with the exception that instead of inactivation with heattreatment, liver homogenates were pre-incubated with approximately 2% Paraoxon 10 minutes prior to the addition of the acetates. Active homogenates and buffer solutions received 40 ul of buffer in place of the Paraoxon.

In Experiment 10, incubation times were decreased to measurements at 0.5, 1, 2.5, 5, and 10 minutes.

Experiment 11 was an attempt to optimize substrate concentrations. The acetates were incubated in 20% liver homogenates at concentrations of 0.025, 0.082, 0.138, 0.194, and 0.250 mM. The samples were processed as described for Experiment 1, and 10 ul were injected onto the HPLC system

Experiment 12 was an attempt to optimize liver homogenate concentrations. The experiment was conducted using n-pentyl and 2-methylbutyl concentrations of 0.25 mM, and liver homogenates in concentrations of 0, 0.2, 1, 5, 10, and 20%. The samples were processed as described for Experiment I, and 10 ul were injected onto the HPLC system.

Experiment 13 was conducted under the assumption that optimal conditions had been achieved. n-Pentyl and 2-methylbutyl acetates in concentrations of 0.05, 0.0832, 0.250, 0.500, and 0.830 mM were incubated with 3% liver homogenates. The samples were processed as described for Experiment 1, and 10 ul were injected onto the HPLC system. It was discovered that essentially all of the acetate had been converted to alcohol at all concentration levels.

Definitive Testing
After optimal experimental conditions were determined, the rates of hydrolysis of the acetates to their corresponding alcohols in F-344 rat liver homogenates were estimated using the following procedures.

Just prior to the start of the experiment, approximately 69 ul of the 20% rat liver homogenate was added to the incubation vials requiring active or
inactive liver homogenate. To this was added 391 ul of 50 mM sodium phosphate buffer (pH 7.4), for a final homogenate concentration of 3% (v/v) in a volume of 460 ul.

Four-hundred and sixty ul of buffer, active 3% liver homogenate, or inactivated 3% liver homogenate were added to appropriately-labeled 1 ml GC
vials. (Note: Liver homogenates were inactivated by the addition of approximately 3% of Paraoxon, 10 minutes prior to incubation with the acetate.
Active homogenates received 15 p1 of buffer in place of the inactivator in order to achieve the same final concentration. Incubations of either
14C-n-pentyl acetate or 14C-2-methylbutyl acetate added to buffer, active liver homogenate, or inactive liver homogenate at concentrations of
approximately 0.050, 0.083, 0.250, 0.500, and 0.830 mM were then made by adding 25 ul of the appropriate spiking solution of each acetate in DMSO.
Upon addition of the acetate, the mixtures were incubated at 37C for 1 minute. The incubation was stopped by the addition of 500 ul of methanol.

For the final experiment (Experiment 14), the liver homogenate concentration was decreased to 0.2%, and incubations were made using both 14C-n-pentyl and 14C-2-methylbutyl acetates. The effect of incubation time on the metabolism of each acetate was evaluated at a single incubation concentration of approximately 0.25 mM, at time intervals of 0, 0.5, 1.0, 2.0, 5.0, and 10 minutes. The effect of substrate concentration on the metabolism of each acetate was evaluated for 1 minute at concentrations of 0.10, 0.20, 0.25, 0.50, and 0.75 mM.
Duration and frequency of treatment / exposure:
Not applicable.
Doses / concentrations
Remarks:
Doses / Concentrations:
Not applicable.
No. of animals per sex per dose:
Not applicable.
Control animals:
no
Positive control:
Not applicable.
Details on study design:
No additional information available.
Details on dosing and sampling:
Sample Analyses
Upon addition of the methanol to the incubations, the samples were centrifuged at approximately 6,000 RPM for about 5 minutes, a portion of the supernatent was transferred to an HPLC vial equipped with a 300 ul Polyspring insert, and 100 ul of the sample was injected onto the HPLC system described above. All study samples were analyzed for the presence of either n-pentyl acetate and n-pentyl alcohol, or 2-methylbutyl acetate and 2-methylbutyl alcohol. The identity of the peaks was confirmed using radiolabeled standards in DMSO. The number of umoles of each acetate remaining in each incubation was calculated based upon the percent of total radioactivity in the sample corresponding to the retention time of the acetate and the specific activity of the radiolabeled standards
Statistics:
No additional information available.

Results and discussion

Preliminary studies:
Analytical Chemistry
The HPLC analysis of standard solutions of 14C-n-pentyl acetate and 14C-n-pentyl alcohol in DMSO yielded single peaks with retention times of
approximately 8.3 and 5.1 minutes, respectively. Analysis of standard solutions of 14C-2-methylbutyl acetate and 14C-2-methylbutyl alcohol
in DMSO yielded single peaks with retention times of approximately 8.3 and 5.1 minutes, respectively. A minimum of 400 dpm (2 x background) was required for reliable and reproducible peak detection.

Radiochemical Purity Determinations
Radiolabeled standards of the acetates were analyzed each time the test substances were used. The standards were analyzed in conjunction with the samples, and radiochemical purities were noted at the time the experiments were run. It should be noted that the measured radioactivity of the test substances was lower than the purity stated by the supplier. Since the major impurities in the test substances were tentatively identified as the proposed metabolic products (corresponding alcohols), the decision was made by the Sponsor to proceed with testing, since the impurities were not expected to interfere with either the metabolism or with the analysis of the test samples.

Preliminary Testing
For initial investigations, a 25 mM concentration was selected, based on the results of other work with similar compounds (Simon, 1985). While the
activity of esterases is believed to be quite vigorous (Frederick, 1992), normally requiring liver homogenate concentrations of only 0.2%, a 20% liver homogenate was selected initially to increase the possibility of detecting metabolism. The approach was to decrease the liver homogenate concentration incrementally, as needed.

The results of Experiment 1 indicated that essentially all of the n-pentyl and 2-methylbutyl acetates had been converted to the corresponding alcohols, indicating that metabolism of these acetates proceeded via hydrolysis. The identity of the acetates and alcohols was confirmed by comparison with authentic 14C-radiolabeled standards, which were analyzed concurrently with the samples. Hydrolysis appeared to occur not only in the active and heat inactivated liver homogenates, but also in the buffer-only incubations. This meant that either the TCA used to halt the reactions was causing acid hydrolysis of the esters, or that nonenzymatic autohydrolysis was taking place.

The results of Experiment 2 indicated that neither acetonitrile nor TCA was a suitable solvent to inhibit the hydrolysis of 2-methylbutyl acetate, as
hydrolysis of the acetate was produced when either TCA or acetonitrile was added, not only in incubations containing liver homogenate and 14C-2-
methylbutyl acetate, but also in incubations containing buffer-only and 14C-2-methylbutyl acetate. It was unclear as to whether the hydrolysis seen was because the enzyme(s) were not inactivated by these solvents or because the change in pH caused the nonenzymatic hydrolysis of this acetate compound. This prompted an investigation of the effect of pH on the autohydrolysis of 2-methylbutyl acetate.

The results of investigations of the effect of pH in Experiment 3 indicated no change in the percent of 2-methylbutyl acetate remaining in any of the
incubations. This indicated that the hydrolysis noted in Experiment 1 was nonspontaneous. It was thought that the buffer used in the previous
n experiment may have been contaminated with active liver homogenate. To confirm that the hydrolysis seen in the active liver homogenates was
enzymatic, a series of experiments designed to investigate inhibition of the enzyme(s) was conducted.

In Experiment 4, a comparison of the effects of TCA and methanol on the inhibition of enzymatic hydrolysis of n-pentyl acetate was made. While hydrolysis was not inhibited by the addition of methanol in active homogenates, the addition of methanol did not produce any hydrolysis in the buffer incubations. However, none of the liver homogenate samples had been inactivated prior to the addition of n-pentyl acetate, so there was no way of knowing if hydrolysis could be inhibited by methanol. Liver homogenate and buffer incubations to which TCA had been previously added showed that all
acetate had been converted to n-pentanol. Experiment 5 was conducted to determine if methanol would inhibit the hydrolysis of the acetates. The results indicated that both active and inactive homogenates showed hydrolysis. However, the extent of hydrolysis in inactivated liver homogenates was about half that of the active homogenates. This indicated that 30-minute heat inactivation at 70°C of the liver homogenate was not adequate to inhibit all enzyme activity. Also, the effect of methanol appeared to have little or no effect on enzyme activity. Experiment 6 was essentially a repeat of
Experiment 5, with the exception that the liver homogenate was inactivated by heating at 70°C for 1 hour. The results of Experiment 6 were nearly identical to Experiment 5. The experiment was repeated once more (Experiment 7) to rule out experimental error. The results of this experiment were inconclusive, as a problem with the HPLC system prevented analysis of the samples. This led to the investigation in Experiment 8 of the use of SDS as an agent to inactivate liver homogenate. The experiment was identical in design to Experiment 6, with the exception that SDS was used in place of heat-treatment to inactivate the liver homogenate. Again, the results were the same as those of the previous three experiments. It became apparent that the enzymes involved required a specific inhibitor to prevent hydrolysis. Personal communication with Dr. C. B. Frederick of Rohm and Haas indicated that the addition of Paraoxon, a nonspecific esterase inhibitor, would inhibit the majority of esterase activity. Experiment 9 had essentially the same experimental design as the previous three experiments, with the exception that instead of inactivation with heat treatment, liver homogenates were pre-incubated with Paraoxon 10 minutes prior to the addition of the acetates. Since information on the amount of Paraoxon required was not provided, it was arbitrarily decided that the amount used would be approximately 2-3% of the 2 ml total incubation volume, and the amount needed would be adjusted later, if necessary. Active homogenates and buffer received an equal volume of buffer in place of the Paraoxon. The results of the experiment indicated that the majority of enzymatic activity was inhibited by the addition of Paraoxon. In addition, it was noted that essentially all hydrolysis in the active homogenates had taken place in the first 5 minutes of the 60-minute incubation.

In Experiment 10, an attempt was made to optimize conditions by decreasing the incubation times to 0-10 minutes. The percent of acetate converted to alcohol was greater than 80% at all sample times, so the method was further refined by adjusting the concentration of acetate in the incubations.

Experiment 11 was conducted to investigate the effect of substrate concentration on enzyme activity. The results showed that a11 acetate was converted to alcohol in active homogenates at all substrate concentrations studied. It was noted at this time that the premise for Experiment 11 was in error, as concentration levels higher than the 25 mM previously used should have been investigated. The cost-prohibitive consequences of the high substrate concentrations that would be required prompted the investigation of the effect of liver homogenate concentration on hydrolysis. Experiment 12 was conducted using a substrate concentration of 0.25 mM in liver homogenates, in concentrations of 0.2 to 20%. The results indicated that 3% liver homogenate was at the approximate midpoint of the linear portion of a curve resulting from a plot of percent acetate remaining versus liver homogenate concentration. At this time, it was believed that optimal conditions had been discovered, and definitive measurement of the rates of hydrolysis could begin. However, upon viewing the data resulting from Experiment 13, in which n-pentyl and 2-methylbutyl acetates ranging in concentration from 0 - 0.25 mM were incubated with 3% liver homogenates, it was discovered that essentially all of the acetate had been converted to alcohol at all concentration levels. This resulted in low estimates of the measured rates of hydrolysis. Increasing the substrate concentration was not feasible, due to the quantity of the substrates available. Instead, the concentration of liver homogenate used was decreased to approximately 0.2%. Having at this point established all experimental parameters, definitive testing could begin.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
not applicable
Details on distribution in tissues:
not applicable
Details on excretion:
not applicable

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
For the final experiment (Experiment 14), the liver homogenate concentration was decreased to 0.2%, and the effect of both substrate concentration and incubation time was determined for both n-pentyl and 2-methylbutyl acetates. The effect of incubation time was evaluated following incubations of approximately 0.0913 mM 14C-n-pentyl acetate and approximately 0.0690 mM of 14C-2-methylbutyl acetate in 2% liver homogenates, and samples were withdrawn at 0.5, 1.0, 2.0, 5.0, and 10 minutes. The results indicated that there was a stoichiometric relationship between the amount of acetate disappearing and the percent of the corresponding alcohol appearing in the incubation. The effect of substrate concentration was evaluated for 1 minute at concentrations of 0.10, 0.20, 0.25, 0.50, and 0.75 mM. Lineweaver-Burk plots of inverse substrate concentration (uM(-1)) versus inverse enzymatic hydrolysis velocity [(umoles of substrate metabolized/mg protein/min)-1] were then constructed. Linear regression of these plots was used to determine the Michaelis-Menten, first-order, metabolic (hydrolysis) rate constant (Km) and the maximum velocity (Vmax) of enzymatic hydrolysis for each isomer. The calculated Km ,and Vmax values for n-pentyl acetate were approximately 484 uM and 0.17 umoles/mg/min, respectively. 2-Methylbutyl acetate had calculated Km and Vmax values of approximately 70 uM and 0.09 umoles/mg/min, respectively.

A limitation of this study was that, other than co-elution with authentic standards, no attempt was made to confirm the identity of the metabolic
products by a more rigorous method (such as mass spectral detection), which would give a greater degree of certainty to the identification of the
metabolites as n-pentyl and 2-methylbutyl alcohols.

Any other information on results incl. tables

No additional information available.

Applicant's summary and conclusion

Conclusions:
The calculated Km and Vmax values for n-pentyl acetate were approximately 484 uM and 0.17 umoles/mg/min, respectively, while 2-methylbutyl acetate had calculated Km and Vmax values of approximately 70 uM and 0.09 umoles/mg/min, respectively.
Executive summary:

Primary amyl acetate (CAS No. 14611-52-0) is a product consisting of a mixture of two, 5-carbon alkyl acetate isomers; n-pentyl acetate (CAS No. 6628-63-7) and 2-methylbutyl acetate (CAS No. 624-41-9). The purpose of this study was to determine if the metabolism of these two isomers of primary amyl acetate in rat liver homogenates proceeded via hydrolysis, and, if so, to determine the Michaelis-Menten, first-order, metabolic (hydrolysis) rate constant for each isomer. For this study, the biological conditions under which these acetate esters are hydrolyzed and the characterization of the major metabolic endproducts were investigated using liver homogenate derived from adult male Fischer 344 rats.

14C-Labeled n-pentyl acetate and 14C-labeled 2-methylbutyl acetate were used in this study. An analytical method for the analysis of 14C-n-pentyl acetate, 14C-2-methylbutyl acetate, 14C-n-pentyl alcohol, and 14C-2-methylbutyl alcohol was developed at BRRC, utilizing high performance liquid chromatography (HPLC) and a radioactive flow detector.

The results of preliminary testing involving the incubation of 25 mM 14C-n-pentyl acetate and 14C-2-methylbutyl acetate in 20% liver homogenates for 0-10 minutes demonstrate that essentially all of the 14C-n-pentyl acetate and 14C-2-methylbutyl acetate had been converted to the corresponding alcohols. This is confirmation that the degradation of the acetates proceeded via hydrolysis. The identities of the acetates and alcohols were confirmed by comparison with authentic 14C-radiolabeled standards, which were analyzed concurrently with the samples. Further experiments were conducted to determine that the hydrolysis of the acetates was enzymatic and to optimize experimental conditions. The hydrolysis of the acetates could be inhibited by the addition of approximately 5-10% Paraoxon, an esterase inhibitor, to the liver homogenate incubation, indicating that the hydrolysis was enzymatic.

The enzymatic nature of the hydrolysis was further demonstrated in experiments in which approximately 0.0913 mM 14C-n-pentyl acetate and approximately 0.0690 mM of 14C-2-methylbutyl acetate were incubated with 0.2% liver homogenate in a

37C water bath, and sampled for various times up to 10 minutes. The samples were then analyzed for the acetate ester and its corresponding alcohol. 14C-n-pentyl acetate, 14C-2-methylbutyl acetate, 14C-n-pentyl alcohol, and 14C-2 -methylbutyl alcohol were used to confirm the retention times of the analytes, and to measure the percent of acetate remaining and the percent of alcohol

appearing in each incubation. The results indicated that there was a stoichiometric relationship between the amount of acetate disappearing and the amount of the corresponding alcohol appearing in the incubation.

For determination of the metabolic rate constant for each isomer, 14C-labeled n-pentyl acetate and 14C-labeled 2-methylbutyl acetate were added to 0.2% homogenates in concentrations ranging from approximately 0.25-250 mM for approximately 1 min in a shaking, 37C water bath. The samples were then n analyzed as described above. Lineweaver-Burk plots of inverse substrate

concentration (uM(-1)) versus inverse enzymatic hydrolysis velocity [(umoles of substrate metabolized/mg protein/min)-1] were then constructed. Linear regression of these plots was used to determine the Michaelis-Menten, first-order, metabolic (hydrolysis) rate constant (Km) and the maximum velocity (Vmax) of enzymatic hydrolysis for each isomer.

The results of this study indicate that the most likely route of metabolic degradation of the two isomers in rat liver homogenates is hydrolysis, and that this breakdown is enzymatic. The calculated Km and Vmax values for n-pentyl acetate were approximately 484 uM and 0.17 umoles/mg/min, respectively, while 2-methylbutyl acetate had calculated Km and Vmax values of approximately

70 uM and 0.09 umoles/mg/min, respectively