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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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
toxicokinetics
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
The study is generally equivalent to 417, but was not specifically conducted as such.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
-Name of test material (as cited in study report): Isobutyric acid, 2,2,4-trimethyl-1,3-pentanediol monoester, mixed isomers
Specific details on test material used for the study:
One tin can containing TX (CAS No. 25265-77-4) was received in good condition at WIL Research on 15-Nov-2012. The material was described as a light brown liquid. The test substance was assigned WIL Research reference no. ARS 010502A and stored at room temperature. The tin can was labeled with the following information:
GMN PO210419 Eastman Texanol™ Ester Alcohol
Lot Number: XK11012931
Expiration Date: NA
The purity of the TX test substance was documented as 99.0%. Retention samples were collected per WIL Research SOPs.


One tin can containing TXIB (CAS No. 6846-50-0) was received in good condition at WIL Research on 15-Nov-2012. The material was described as a light brown liquid. The test substance was assigned WIL Research reference no. ARS 010501A and stored at room temperature. The tin can was labeled with the following information:
GMN PO131520 Eastman TXIB™ Formulation Additive
Lot Number: TD11027330
Expiration date: NA
The purity of the TXIB test substance was documented as 99.05%. Retention samples were collected per WIL Research SOPs.
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:

ANIMAL RECEIPT AND ACCLIMATION
Forty-four male Sprague Dawley rats were received at WIL Research on 04-Dec-2012 from Charles River Laboratories (Raleigh, NC). Each rat was inspected by a qualified technician upon receipt. Rats were judged to be in good health and immediately placed in acclimation for approximately 1 week. All rats were weighed and uniquely identified by a metal ear tag displaying a permanent animal number. During the acclimation period, each rat was observed twice daily for changes in general appearance or behavior.

ANIMAL HOUSING
All animals were housed individually in clean, suspended wire-mesh cages in an environmentally controlled room. Cages were elevated above cage-board, which was changed at least 3 times each week. Individual cage cards were affixed to each cage displaying the animal number, group number, and study number.

DIET AND DRINKING WATER
PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was offered ad libitum during acclimation and the biological phases of the study. WIL Research SOPs provide specifications for acceptable levels of heavy metals and pesticides that are reasonably expected to be present in the diet without interfering with the purpose or conduct of the study. The feed lots used during the study were documented in the study records. Reverse osmosis-purified water was provided ad libitum. Water was provided using an automatic system. The municipal water supplying the laboratory is analyzed for contaminants according to WIL Research SOPs to ascertain that none of the analyzed contaminants were present in concentrations that would be expected to affect the outcome of the study.

ENVIRONMENTAL CONDITIONS
Environmental controls were set to maintain an average daily temperature of 71°F ± 5°F (22°C ± 3°C) and an average daily relative humidity of 50% ± 20%. Temperature and relative humidity were monitored continuously. Data for these 2 parameters were scheduled for automatic data capture on an hourly basis. Animals were housed in room B-121. Actual daily mean temperatures ranged from 69.8°F to 71.0°F (21.0°C to 21.7°C) and actual daily mean relative humidity ranged from 34.9% to 41.4%. Timers were set to provide a daily photoperiod of 12 hours of fluorescent light followed by 12 hours of darkness. The ventilation rate was set at a minimum of 10 air changes per hour, 100% fresh air.

Administration / exposure

Route of administration:
other: IV and oral gavage for individual phases
Vehicle:
other: 50/50 propylene glycol:ethanol
Details on exposure:
SELECTION PROCEDURES AND GROUP ASSIGNMENT
Near the end of the acclimation period, animals were judged to be suitable for testing, weighed, and assigned to groups at random using a computer program. Animals that were selected for IV administration (Groups 1 and 3) were used for the collection of whole blood for the in vitro metabolism phase.

Dose administration was initially performed on 11-Dec-2012 by intravenous injection to Groups 1 and 3. However, due to the unexpected expiration of 3 of 4 animals in Group 1 and 1 of 1 animal in Group 3 immediately following test substance administration, dosing was stopped and the study was postponed to a later date. Groups 1 and 3 were permanently discontinued. Samples collected from animals that expired were not analyzed.

Dosing was resumed on 04-Jan-2013 with administration of the oral dose. Animals were weighed on the day of dosing. Based on these weights, the dose to be administered was calculated on a mg/kg body weight basis using the dosage volume of 5 mL/kg. Animals weighed between 323 and 443 g and were approximately 11 weeks of age at initiation of dosing. Each animal in Group 2 was administered a single oral (gavage) dose of TX at 100 mg/kg. Each animal in Group 4 was administered a single oral (gavage) dose of TXIB at 100 mg/kg. The dose formulations were administered within 36 hours of dose preparation
Duration and frequency of treatment / exposure:
See "details on dosing and sampling"
Doses / concentrations
Remarks:
See "dosing and sampling"
No. of animals per sex per dose:
See "dosing and sampling"
Control animals:
no
Positive control:
no
Details on study design:
See "dosing and sampling"
Details on dosing and sampling:
PARAMETERS EVALUATED

EVALUATION OF TEST SUBSTANCE SUITABILITY
To evaluate the selected vehicle for acceptable use in the study, 2 male rats were weighed and administered a single 1 mL/kg IV (bolus) dose of vehicle (PG EtOH [50:50]) based on their weight. No adverse effects were noted and the vehicle was considered acceptable for IV (bolus) dosing at the indicated volume by weight. No samples were collected from the rats for analysis.

PHARMACOKINETIC PHASE
BLOOD COLLECTION AND PROCESSING
At approximately 5, 10, 15, 30, 45, and 60 minutes post-dosing, approximately 1.0 mL of blood was obtained via the jugular vein from 5 rats/sub-group (alternating between 2 subgroups per time-point) and transferred to a chilled blood collection tube containing sodium heparin as the anticoagulant. The final blood collection was terminal; animals were euthanized by inhalation of CO2 and as much blood as possible was collected from the vena cava.
Blood samples were centrifuged within 30 minutes of collection under refrigerated conditions to isolate plasma. The plasma samples were stored at approximately -70°C until transferred to the WIL Research Bioanalytical Chemistry Department for analysis of the indicated analytes using validated LC-MS methods (Nethero, 2016, WIL-387043). Following the terminal blood collection, the carcasses were discarded.

IN VITRO METABOLISM PHASE
BLOOD COLLECTION AND PROCESSING
Ten animals were euthanized by inhalation of CO2 and a whole blood sample (as much blood as possible) was collected from the vena cava via syringe and immediately transferred to a blood collection tube containing sodium heparin as the anticoagulant. Following the terminal blood collection, the carcasses were discarded.
Five of the collected whole blood sample tubes received 250 μL of 100 mg/mL TX in 50:50 PG:EtOH (reference no. 387037MA1-9-1) and the remaining 5 whole blood sample tubes received 250 μL of 100 mg/mL TXIB in 50:50 PG:EtOH (reference no. 387037MA1-10-1). The tubes were capped and gently mixed with inversion. Using a pipette, 1 mL of each of the spiked blood samples were placed into 6 appropriately-labeled glass vials for 0, 5, 10, 15, 20, and 30 minute incubations of each sample. The vials were capped and placed on a rocking platform to incubate for the appropriate amount of time at approximately 37°C. At each appropriate time-point, 1 mL of ice-cold EtOH was added to each vial and then placed on ice for a minimum of 20 minutes. Samples were then centrifuged for 20 minutes at approximately 2500 × g under refrigerated conditions to precipitate protein. The supernatant fraction of each sample was transferred into an appropriately-labeled glass vial and placed into storage at approximately -70°C until transfer to the WIL Research Bioanalytical Chemistry Department for analysis of the indicated analytes. Qualified LC-MS methods were also used for the analysis of the precipitated whole blood supernatant fraction.
Statistics:
CALCULATIONS
Calculations for this report were performed in Microsoft Excel® 2007 spreadsheets using full
floating point decimals. Equations other than for the mean, SD, or CV% are presented with the
tables. Slightly different results can be expected if calculations are based on the values as
presented in the tables because some numbers have been rounded for display.

STATISTICS

All statistical tests were performed using WTDMS™. Data was rounded to 2 decimal places for
importation into WTDMS™. Analyses were conducted using a Student’s two-sample t-test (Sokal and
Rohlf, 1981) at significance level of 5%, comparing each analyte within each incubation relative to
time 0. Values under range (statistical analysis and reporting.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
See "overall remarks, attachments"
Details on distribution in tissues:
See "overall remarks, attachments"
Details on excretion:
See "overall remarks, attachments"

Metabolite characterisation studies

Details on metabolites:
See "overall remarks, attachments"

Bioaccessibility

Bioaccessibility testing results:
See "overall remarks, attachments"

Applicant's summary and conclusion

Conclusions:
Neither TX nor TXIB appear to be absorbed into systemic circulation, as indicated by the absence of TX, TXIB, TMPD, or isobutyric acid in plasma following a single oral dose of TX or TXIB.
In vitro experiments in whole blood fortified with TX and TXIB indicate that TX is stable for up to 30 minutes at 37°C in fresh whole blood, whereas TXIB appears to undergo limited hydrolysis. However, although there was a small but statistically significant increase in the concentration of potential metabolites of TX and TXIB (TMPD and IBA), the concentration never exceeded 4.5% of the starting test substance concentration in any of the incubations.
Executive summary:

OBJECTIVE

The objective of this study was to determine the plasma pharmacokinetics of TX, TXIB, and metabolites in male Sprague Dawley Crl:CD(SD) rats following the administration of a single oral (gavage) dose of either TX or TXIB. The second objective was to determine the rate and extent of hydrolysis of TX and TXIB in freshly-collected whole blood fortified with TX orTXIB.

 

STUDY DESIGN

For the pharmacokinetic phase of the study, 2 dose groups received a single oral (gavage) dose of TX or TXIB at 100 mg/kg. Each group consisted of 10 male rats. Blood samples were collected from 5 rats/time point alternating between 2 sub-groups at 6 time points up to 1 hour post-dosing. Plasma samples were analyzed for TX (1- and 3-substituted), TMPD, and isobutyric acid (group receiving TX) or TXIB, TX (1- and 3-substituted), TMPD, and isobutyric acid (group receiving TXIB).

For thein vitrometabolism phase, whole blood was collected from male rats via the vena cava. A fixed aliquot of TX or TXIB was added to each whole blood sample and incubated in closed vials. The starting concentration of TX varied based on the volume of blood collected; the measured concentration at time zero ranged from 2040 μg/mL to 3310 μg/mL. Similarly, the starting concentration of TXIB ranged from 2290 μg/mL to 3640 μg/mL. At 6 time points up to 30 minutes post-dosing, enzymatic activity was quenched by the addition of ice-cold EtOH. Samples were analyzed for TX (1- and 3-substituted), TMPD, and isobutyric acid (TX incubations) or TXIB, TX (1-and 3-substituted), TMPD, and isobutyric acid (TXIB incubations).

 

RESULTS AND DISCUSSION

Following oral administration of TX or TXIB to male rats at 100 mg/kg, the concentrations of TX (1- and 3-substituted), TXIB, TMPD, and isobutyric acid in rat plasma were all below the lower limit of quantitation for the analytical method (<5 μg/mL) at 5, 10, 15, 30, 45, and 60 minutes post-dosing.

The concentration of TX in whole blood fortified with TX was unchanged over the time course (up to 30 minutes at 37°C). There appeared to be a small increase in the concentration of TMPD, from 15.6 μg/mL at 0 minutes to 115 μg/mL after 30 minutes of incubation, but even after 30 minutes, it was equivalent to <5% of the starting TX concentration. There also appeared to be a small increase in the concentration of IBA, from BLQ at 0 minutes to 48.4 μg/mL after 30 minutes of incubation.

The concentration of TXIB in whole blood stayed consistent (89.5% to 99.8% of the starting concentration) over the first 20 minutes of incubation, with no apparent trend in concentration over time. After 30 minutes of incubation, the concentration of TXIB was 82.4% of the starting concentration. The concentration of TX in the same samples increased steadily from 15.4 μg/mL (0.54% of the TXIB concentration) at the beginning of the incubations, to 55.6 μg/mL (2.0% of the starting TXIB concentration). No TMPD was detected in the TXIB whole blood incubations. The concentration of IBA in TXIB whole blood incubations was BLQ until 20 minutes post-dosing, but the maximum mean concentration at any time point was <25 μg/mL.