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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable with restrictions; in vitro rather than in vivo study using low numbers of animals. The study was conducted according to GLPs.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Deviations:
not applicable
Principles of method if other than guideline:
Naïve male Sprague-Dawley rats were anesthetized and 5-10 mL of blood was withdrawn by cardiac puncture. All blood samples were immediately transferred to Vacutainer™ tubes containing sodium heparin. Venous blood was withdrawn from two male volunteers using Vacutainer™ tubes and collecting systems. All blood was used immediately for hydrolysis testing. Aliquots of blood (4.95 mL) were placed into pre-warmed (37 °C) Erlenmeyer flasks in a gyratory water bath and concentrated solutions of Texanol (50 µL) in dimethyl sulfoxide (DMSO) were added to initiate the hydrolysis studies. The samples were shaken and a 0.5 mL sample was immediately removed for extraction and gas chromatographic analysis. Samples were removed at appropriate intervals up to a maximum of 2 hours. In a separate study, Texanol in DMSO was added to phosphate buffer; aliquots were sampled at 0, 60 and 120 minutes, and analyzed as the other samples.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Test substance:
-Test substance (as cited in report): 2, 2, 4-Trimethyl-1, 3-Pentanediol Monoisobutyrate (Texanol)
-Source of test material: Eastman Chemical Company, Kingsport, TN
-Purity: 99.9%
-Method of Analysis: performed by Chemical Quality Services Division, Eastman Kodak Company, Rochester, NY. Analysis was by gas chromatography-mass spectrometry (GC-MS). Texanol is a mixture of the 1-substituted and 3-substituted monoisobutyrate isomers of TMPD consisting of 64.5% of the 1-substituted isomer and 35.5% of the 3-substituted monoester.

Test substance:
-Test substance (as cited in report): 2, 2, 4-trimethyl-1, 3-pentanediol (TMPD)
-Source of test material: Eastman Chemical Company, Kingsport, TN
-Purity: 99.3%
-Method of Analysis: performed by Chemical Quality Services Division, Eastman Kodak Company, Rochester, NY. Analysis was by GC-MS.
Radiolabelling:
no

Test animals

Species:
other: Rat and Human
Strain:
other: Sprague-Dawley rat and Human
Sex:
male
Details on test animals and environmental conditions:
-Strain: Sprague-Dawley rats [Crl:CD®-(SD)Br] (male) and humans (male)
-Source of rats: Charles River Laboratories, Kingston, NY
-Source of human males: personnel in the Biochemistry Unit, Eastman Kodak Company, Rochester, NY
-Feed for rats: Certified Rodent Diet (Agway Prolab RMH 3000) ad libitum
-Human diet prior to study: unknown
-Water for all test subjects: Rochester local water municipal ad libitum
-Identification: rats were identified with a uniquely numbered ear tag
-Weight of rats at study termination: 200-250 grams

Administration / exposure

Route of administration:
other: in vitro study
Vehicle:
DMSO
Details on exposure:
Animals and humans were not treated directly with test substances. Test subjects were used for blood collection purposes only. Hydrolysis studies were carried out on isolated rat and human blood samples.
Duration and frequency of treatment / exposure:
Single aliquots of Texanol in DMSO were added to blood samples or phosphate buffer; samples removed at set intervals over a 2 hour period.
Doses / concentrations
Remarks:
Doses / Concentrations:
Nominal concentrations of Texanol in blood were 4.6 and 2.3 mM; nominal concentration in buffer was 1.35 mM.
No. of animals per sex per dose:
Rats: 3 males; Humans: 2 males
Control animals:
no
Details on study design:
Rats (n=3) were anesthetized with methoxyflurane and 5-10 mL of blood was withdrawn by cardiac puncture. All blood samples were immediately transferred to Vacutainer™ tubes containing sodium heparin. Venous blood was withdrawn from two male volunteers using Vacutainer™ tubes and collecting systems. The blood was used immediately for hydrolysis testing. Aliquots of blood (4.95 mL) were placed into pre-warmed (37 °C) Erlenmeyer flasks in a gyratory water bath and concentrated solutions of Texanol (50 µL) in DMSO were added to initiate the hydrolysis studies (final concentrations of Texanol of 4.6 and 2.3 mM). The samples were shaken and a 0.5 mL sample was immediately removed for extraction and gas chromatographic analysis. The flasks were returned to the water bath and samples were removed at appropriate intervals (0, 5, 10, 20, 30, 45, 60, 90, and 120 minutes). In a separate control experiment, a DMSO solution of Texanol was added to a 4.95 mL aliquot of 0.1M pH 7.0 phosphate buffer pre-warmed to 37 °C (nominal concentration of 1.35 mM). The samples were shaken and 0.5 mL samples were removed for extraction and gas chromatographic analysis at the appropriate intervals (0, 60, 120 minutes).

Prior to gas chromatographic analysis, the blood samples (0.5 mL) were added to culture tubes containing 1.0 mL of 15% aqueous zinc sulphate solution, 1.0 mL of ethyl acetate (containing 2.0 mM n-octanol as an internal standard), and a 1.6 mL aliquot of saturated aqueous barium hydroxide. The tubes were shaken, centrifuged and the top organic phase was removed and dried by passing through anhydrous sodium sulphate packed within a disposable pipette plugged with glass wool. To ensure complete transfer, an additional 1.5 mL of ethyl acetate was added to each culture tube as a wash and this second organic phase was removed and filtered as above. The dried extracts were collected for each sample and the final volume adjusted to 2.0 mL with ethyl acetate. Chromatographic standards of Texanol and TMPD in DMSO were also prepared, extracted, and analyzed by gas chromatography. All extracts were analyzed with a Flame Ionization gas chromatograph. Quantitation of both Texanol and TMPD was accomplished using the extracted standards and n-octanol as an internal standard. Individual sets of data were analyzed by a simple linear regression analysis.
Statistics:
Standard statistical analyses of the hydrolysis data were used where appropriate.

Results and discussion

Any other information on results incl. tables

At a nominal initial starting concentration of 4.6 mM Texanol, gas chromatographic analyses of whole rat blood indicated a concentration of 3.6 mM (summation of both isomers) at 0 min or 78.4% of the nominal value. Loss of the 1-substituted isomer was non-linear for the first 10 minutes and the 1-substituted isomer decreased in concentration from 2.3 mM to 1.3 mM in 30 minutes whereas the concentration of TMPD increased to 1.1 mM in 30 minutes. The concentration of 3-substituted isomer remained unchanged during this time. At a nominal initial starting concentration of 2.3 mM Texanol, the analyzed concentration of Texanol isomers at 0 min was equivalent to 82.9% of the nominal dose. In addition, TMPD was equivalent to 83.8% of the hydrolyzed Texanol following a 30 min incubation. Linear regression analysis provided a half-life of 21.8 minutes for loss of the 1-substituted isomer. In a second study with rat blood, the initial Texanol concentration as determined by GC analysis was equivalent to 88.7% of the nominal initial starting concentration of 2.0 mM. Linear regression analysis provided a half-life of 17.4 minutes. 

Analyzed concentrations of Texanol in human blood were between 72.3-79.2% of the nominal dose (~2 mM) at 0 minutes and linear regression analysis provided a half-life of 15.7-18.9 minutes for the loss of the 1-substituted isomer. The TMPD produced was equivalent to 78.5- 95.4% of the hydrolyzed Texanol. 

In the control study conducted in phosphate buffer rather than blood, Texanol in DMSO at the 0 min analysis resulted in a Texanol concentration of 1.2 mM or 90.5% of the nominal dose (1.4 mM). Samples analyzed at approximately 60 and 120 minutes gave Texanol concentrations of about 1.2 mM, not significantly different from the 0 min measurement. TMPD concentrations did not increase over the course of the study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: In vitro, Texanol undergoes partial hydrolysis in whole rat or human blood to produce the expected diol, TMPD. Approximately 35.5% of the starting material did not undergo hydrolysis in either rat or human blood under conditions of this study.
In an in vitro metabolism study in which different concentrations of Texanol were exposed to whole rat or human blood, the 1-substituted isomer, which is the major component of Texanol (64.5%), undergoes partial hydrolysis to produce the expected diol, TMPD. The 3-isomer of Texanol, which is the minor component (35.5%), did not undergo hydrolysis in either rat or human blood. Hydrolysis of the 3-isomer may occur in other tissues of the body but this possibility was not investigated in this study. Based on the results of these in vitro studies performed in isolated blood, the hydrolytically stable 3-isomer would be expected to have a different pharmacokinetic profile from that of the corresponding 1-isomer.
Executive summary:

In a series of in vitro metabolism studies, naïve samples of blood isolated from humans and rats were exposed to different concentrations of 2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate (Texanol), a mixture consisting of 64.5% of the 1-substituted isomer and 35.5% of the 3-substituted isomer. The purpose of the studies was to determine the rate and extent of hydrolysis of Texanol to TMPD in whole blood samples at 37 °C. Two hydrolysis studies were conducted in rats and a study was also performed using blood from two human subjects. In the first rat study, two initial nominal concentrations of Texanol were used. Loss of the ester at the higher concentration (4.6 mM) did not follow first-order kinetics. At this concentration level, either the water solubility of the ester had been exceeded, or saturation of the blood esterase enzymes may have occurred. At the lower concentration level (2.3 mM), loss of the 1-substituted isomer followed first-order kinetics with a calculated half-life of 21.8 minutes. This lower concentration study was repeated (nominal concentration 2.0 mM) and yielded a first-order half-life of 17.4 minutes. The average half-life for the 1-substituted isomer under conditions of this study was 19.6 minutes. Half-lives for the hydrolysis in human blood of the 1-substituted isomer were 15.7 and 18.9 minutes for the two trials, giving an average of 17.3 minutes. The 3-substituted isomer of Texanol was resistant to hydrolysis under the conditions used in these studies. For both species, the single identified product resulting from hydrolysis of the 1-substituted isomer was TMPD. The results of these in vitro studies suggest that the 1-substituted and 3-substituted isomers of Texanol may differ in their metabolic uptake, distribution, metabolism, and elimination.