2,6,8-trimethylnonan-4-ol

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

circa 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to guidelines but was conducted according to GLPs and the report contains sufficient data for interpretation of study results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Objective of study:

other: pharmacokinetics and metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study four male rats were administered either IBHK or TMN alcohol (TMN) at nominal concentrations of 100 mg test material/kg bw via single oral gavage. Plasma and RBC time-courses were conducted and excreta collected for 168 hours post-dosing. Selected tissues were collected at sacrifice and analyzed for radioactivity. Similarities and/or differences in pharmacokinetics and metabolism were evaluated via comparison of absorption, distribution, and excretion kinetics and metabolite profiles. These data were utilized to compare bioequivalence for these two compounds in the male Crl:CD(SD) rat.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Details on test material:

Two structurally similar chemicals were studied.

1) Isobutyl heptyl ketone, (synonym IBHK, 2,6,8-Trimethyl-4-nonanone, 2,4,8-Trimethylnonan-6-one)
Non-Radiolabeled:
The Dow Chemical Company c/o Chemical Marketing Concepts, New Milford, Connecticut (Lot # VC1555T629, Plant Quality Coordinator (2007)).
Radiolabeled:
Tjaden Biosciences, Burlington, Iowa (INV TJBIOS-NB14-77-ketone)
Non-radiolabeled purity: The GLP certificate of analysis lists the purity of the test material as 96.8% by weight. There were six impurities detected >0.1% and ranged from 0.110 to 1.12%. The water content was 0.04%.
Radiochemical purity: 98.6% (Agilent 1100 HPLC system with a Berthold LB 509 flow RAM detector). Position of (14)C-IBHK (2,6,8-Trimethyl-4-nonanone, [3-(14)C]) Specific Activity of Radiolabeled Test Material = 20 mCi/mmol.

2) 2,6,8-Trimethyl-4-nonanol
Non-Radiolabeled
The Dow Chemical Company, Freeport, Texas (Lot # AA0155T32V, Plant batch record (2008)).
Radiolabeled
Tjaden Biosciences, Burlington, Iowa (INV TJBIOS-NB14-77-alcohol)
Non-radiolabeled purity: The GLP certificate of analysis lists the purity of the test material as 98.0% by weight. There were two impurities detected >0.1% and ranged from 0.152 to 1.45%. The water content was 0.05% .
Radiochemical purity: 99.4% (Agilent 1100 HPLC system with a Berthold LB 509 flow RAM detector). Position of (14)C-TMN alcohol (2,6,8-Trimethyl-4-nonanol, [3-(14)C]). Specific Activity of Radiolabeled Test Material = 20 mCi/mmol.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Male Crl:CD(SD) rats were selected because of their use in previous toxicity studies. Rats are suitable species for the analysis of metabolism of chemicals in vivo. The Crl:CD(SD) rat was also suitable due to the availability of historical background data, and the reliability of the commercial supplier.

Supplier and Location
Taconic (Germantown, New York)

Age at Study Start
9 weeks

Physical Acclimation
Upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International), each animal was evaluated by a laboratory veterinarian to determine the general health status and acceptability for study purposes. Jugular vein cannulated rats (surgery performed by the supplier) were acclimated in metabolism cages for approximately five days prior to
use.

Housing
Following administration of test material the animals were housed singly in glass Roth-type metabolism cages, which were designed for the separation and collection of urine, feces, CO2, and organic volatiles. Air was drawn through the metabolism cages at ~ 850 mL/minute.
Temperature: 22 °C with a tolerance of ± 1 °C (and a maximum permissible excursion of ± 3 °C)
Humidity: 40-48%
Air Changes: 12-15 times/hour

Randomization and Identification
The jugular vein cannulated rats were stratified by body weight and/or patency of their cannulae, and then randomly assigned to treatment groups using a computer program. Animals placed on study were uniquely identified with alphanumeric metal ear tags.

Feed and Water
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed and municipal water was provided ad libitum except that access to feed was restricted approximately 16 hours prior to the administration of test material and was returned about 4 hours post-dosing. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.

Cannulation
Jugular Vein
Rats were obtained already cannulated in the jugular vein by the supplier.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Rats were orally dosed with radiolabeled IBHK or TMN alcohol at a nominal concentration of 100 mg/kg and excreta collected (Table 1). Time-course blood was collected (e.g., 5, 10, 15, 30 minutes and 1, 2, 3, 6, 12, 24, and every 24 hours post-dosing thereafter from each animal). The study continued for seven days post-dosing.

Oral Dose Administration
The oral dose was administered via a ball-tipped gavage needle attached to a glass syringe. Animals from Groups 1 and 2 received a single oral dose. The volume of radiolabeled dose formulation administered to each animal was calculated based on the body weight taken on the day of dose administration. The actual amount of radiolabeled test material administered to each animal was determined by weighing the dose syringe before and after dose administration. The targeted volume of the radiolabeled dose formulation was 5 g/kg bw. Groups 1 and 2 animals had restricted feed overnight through approximately four hours post-dosing.

Route and Justification, Method of Administration, Frequency and Duration
A possible route of human exposure to the test material was via residues in food. Thus, oral administration to rats via oral gavage represented an appropriate means of exposure. Traditionally, kinetic analysis of a chemical was conducted following either oral or IV bolus dosing. The best way to determine the unbiased pharmacokinetics of any chemical was after administering bolus IV dose; however, this was not relevant for the test material whose primary route of exposure was via residual level in food. Therefore, determining the kinetic parameters after oral gavage dosing was more appropriate. Bolus oral gavage was also to determine the exact dose administered which would not be possible if provided in diet.

Dose Levels and Justification
A dose of 100 mg/ bw was used in this study, which was high enough to provide sufficient levels in excreta that were easily be subjected to chemical analysis.

Dose Preparation
Oral Dose Preparation
The 14C-IBHK suspension was prepared by adding 144 μL of IBHK and 600 μCi of 14C-IBHK (dissolved in ethanol) into a 20-mL glass vial containing a magnetic stir bar. The contents were stirred until well mixed. This mixture was evaporated to dryness using a gentle stream of nitrogen. Approximately 5.9 mL of corn oil vehicle was added to the vial and the contents were stirred until the chemical was dissolved in the vehicle. This suspension was diluted to volume for a final target concentration of 21.74 mg/g (100 μCi/mL) and target radioactivity level of 109 μCi/g. The 14C-TMN alcohol suspension was prepared by adding 142 μL of TMN alcohol and 600 μCi of 14C-TMN alcohol (dissolved in ethanol) into a 20-mL glass vial containing a magnetic stir bar. The contents were stirred until well mixed. This mixture was evaporated to dryness using a gentle stream of nitrogen. Approximately 5.9 mL of corn oil vehicle was added to the vial and the contents were stirred until the chemical was dissolved in the vehicle. This suspension was diluted to volume for a final target concentration of 21.74 mg/g (100 μCi/mL) and target radioactivity level of 109 μCi/g.

Target dose volume was 5 g/kg body weight, target dose amount was 100 mg/ body weight and target radioactivity was 500 μCi/kg body weight. Confirmation of the test material concentration in the dose suspensions was conducted. Radioactivity in the dose suspensions were quantified by liquid scintillation spectrometry (LSS) as described below.

Analysis
Dose Confirmation and Homogeneity of Dose Suspension
The concentration of IBHK and TMN alcohol in the respective dose suspensions were conducted in accordance with the standard operating procedures of the Analytical Chemistry Laboratory. Dose confirmations were performed using gas chromatography with flame ionization detection (GC/FID). Specific details of the analytical method were included in the study file. LSS analysis of aliquots of the 14Clabeled dosing suspensions taken from various locations in the suspension container was used to confirm the concentration of radioactivity and the homogeneity of the 14C-IBHK and 14C-TMN alcohol in the dosing suspensions.

Stability
Stability of the IBHK was determined to be 28 days at concentrations ranging from 2.5 to 25.0 mg/mL in corn oil. Stability of the TMN alcohol was determined to be 107 days for 0.025 to 250 mg/mL in corn oil.

Duration and frequency of treatment / exposure:

Groups of 4 male rats received a single oral gavage dose of 100 mg/kg.

Doses / concentrations

Remarks:

Doses / Concentrations:
100 mg/kg of 14C-IBHK or 14C-TMN alcohol.

No. of animals per sex per dose / concentration:

Groups of 4 male rats received IBHK or TMN alcohol.

Control animals:

not specified

Positive control reference chemical:

Not applicable.

Details on study design:

Time-Course Plasma and RBC Collection
The rats of Groups 1 and 2 were fitted with indwelling jugular vein cannulae and plasma and red blood cell (RBC) 14C concentration-time course was evaluated (Text Table 1). Blood (~ 0.1-0.2 mL) was collected at 5, 10, 15, 30, minutes and 1, 2, 3, 6, 12, 24, and every 24 hours post-dosing thereafter from each animal and processed for the determination of radioactivity. Plasma was weighed and radioactivity determined by LSS. Weighed aliquots of RBC were oxidized and analyzed for radioactivity by LSS.

Details on dosing and sampling:

Oral Dose Administration
The oral dose was administered via a ball-tipped gavage needle attached to a glass syringe. Animals from Groups 1 and 2 received a single oral dose. The volume of radiolabeled dose formulation administered to each animal was calculated based on the body weight taken on the day of dose administration. The actual amount of radiolabeled test material administered to each animal was determined by weighing the dose syringe before and after dose administration. The targeted volume of the radiolabeled dose formulation was 5 g/kg bw. Groups 1 and 2 animals had restricted feed overnight through approximately four hours post-dosing.

Specimen Collection
Urine
All urine voided during the study was collected in dry-ice cooled traps. The urine traps were changed at 12 and 24 hours post-dosing followed by 24-hour intervals for the remainder of the study. The cages were rinsed with water at the time the traps were changed and the rinse collected. Each urine specimen and urine/cage rinse was weighed, and a weighed aliquot of each sample was analyzed for radioactivity by LSS as described below. Equal volume aliquots of urine samples (per time and test material) from the 0-12-hour, 12-24-hour and 24-48 hour collection intervals was pooled and stored at –80 ºC and selected urine samples underwent chemical analysis.

Feces
Feces were collected in dry-ice chilled containers at 24-hour intervals. An aqueous homogenate (~ 25% w/w) was prepared (shaken for >4 hours) and weighed aliquots of these homogenates was oxidized, (OxiMate 80 Sample Oxidizer, PerkinElmer Life Sciences, Inc., Boston, Massachusetts) and quantitated for radioactivity by LSS. In addition, equal volume aliquots of fecal homogenates from each animal were taken from the 0-24-hour and 24-48-hour collection intervals and pooled (per time and test material). These pooled samples were stored at -80ºC and selected fecal samples underwent chemical analysis.

Expired Volatiles
Air was drawn through the cage at approximately 850 mL/minute. Upon exiting the cage, the air was passed through charcoal or other appropriate trapping agent to trap expired volatiles. The charcoal traps were changed at 24-hour intervals. The charcoal was ground with a blender. Weighed aliquots of the charcoal was oxidized and analyzed for radioactivity by LSS. Because <1% of the administered dose (average per group) was detected in the charcoal trap during the 24-hour interval, the replacement traps were not analyzed for radioactivity. One extra rat for each test material was dosed and placed in the Roth cage to capture expired volatiles to determine if the definitive study recovered all radioactivity due to the lid being opened several times throughout the blood time-course sampling. This extra animal data (not shown) was similar to the radioactivity recovered in the expired volatiles of the definitive study, indicating that the removal of the lid was not a factor in its recovery.

Expired CO2
Following the charcoal trap (described above) the expired air was passed through a solution of monoethanolamine:1-methoxy-2-propanol (3:7 v/v) to trap expired CO2 and analyzed for radioactivity by LSS. The CO2 trap was changed at 12-hour and 24-hour intervals. Because <1% of the administered dose was detected in the CO2 trap during the 24-hour interval, the replacement traps were not analyzed for radioactivity. One extra rat
for each test material was dosed and placed in the Roth cage to capture expired CO2 to determine if the definitive study recovered all radioactivity due to the lid being opened several times throughout the blood time-course sampling. This extra animal data (not shown) was similar to the radioactivity recovered in the expired CO2 of the definitive study, indicating that the removal of the lid was not a factor in its recovery.

Terminal Sacrifice
At 168 hours post-dosing the animals were anaesthetized with CO2/O2 and sacrificed by exsanguination. Following sacrifice the Roth cages were washed and the cage wash analyzed for radioactivity.

Tissues
The following tissues were collected from each group at sacrifice:
Plasma
RBC
Carcass
GI tract
Kidney
Liver
Skin
Thyroid
Testis
Blood was collected and centrifuged to separate plasma and RBC. Plasma was analyzed for radioactivity by LSS. RBC were oxidized and analyzed for radioactivity by LSS. The carcass, GI tract with contents, kidney, liver, and testis were collected, homogenized (~ 33% homogenate), a weighed aliquot oxidized, and analyzed for radioactivity by LSS.

The skin was removed from the carcass and a representative skin sample oxidized and analyzed for radioactivity by LSS. The remaining tissues were directly oxidized without homogenization and analyzed for radioactivity by LSS.

Final Cage Wash
Following the terminal sacrifice of the animals, a final cage wash (FCW) was performed. The final cage wash and contents were collected and the weight of the sample was determined. The FCW and contents were shaken for >4 hours. A weighed aliquot of the final cage wash was analyzed for radioactivity by LSS.

Blood (Plasma and RBC)
Blood was obtained at sacrifice via cardiac puncture. Blood was collected into tubes containing sodium heparin anticoagulant from all animals at sacrifice and separated into plasma and RBCs.

Control Samples
Control urine (12- and 24-hours) and feces (24-hours) were collected in dry-ice cooled traps from one male dosed with the corn oil vehicle containing no test material. Samples were stored at -80 °C for chemical analysis.

Sample Analysis
14C Analysis
Radioactivity was quantified in a liquid scintillation spectrometer (Packard Tri-Carb 2900TR). Counts per minute (cpm) were corrected for quench and converted to disintegrations per minute (dpm). Samples with dpm less than twice the concurrently run background were considered to contain insufficient radioactivity to reliably quantify (less than the limit of quantitation; those samples were denoted with an “NQ” in the Results Tables). Descriptive statistics were conducted, (i.e., mean ± standard deviation [± SD]). Non-quantifiable excreta samples (urine/rinse/feces) are identified in the tables as NQ and a value of '0' used in calculations. For tissues, when a sample was non- quantifiable, that sample was assigned the quantitation limit (QL)
for calculations and displayed as NQ with the quantitation limit in parenthesis. The mean is calculated from actual values and calculated QL values and presented as ± SD, unless greater than ½ of the values are presented as NQ, in which case the mean is expressed as NQ () ± SD. If all tissue values are NQ the mean is presented as NQ (QL) with no SD displayed.

Metabolite Profiles
Pooled samples (Groups 1, and 2; urine and feces) from selected intervals were analyzed for parent and metabolite(s) via HPLC with in-line radiochemical detection.

Statistics:

Descriptive statistics were used, i.e., mean ± standard deviation. All calculations in the database were conducted using Microsoft Excel spreadsheets and databases in full precision mode (15 digits of accuracy). Certain pharmacokinetic parameters were estimated for plasma and RBC data, including AUC (area-under-the-curve), Cmax and elimination rate constants, using the pharmacokinetic computer modeling program PK Solutions (v2.0.6.; Summit Research Services, Montrose, Colorado).

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Absorption of Orally Administered Dose
Absorption of the oral doses was estimated from the percent of administered radioactivity recovered in urine and rinse, final cage wash, CO2 and tissues at sacrifice (Table 2). The total absorption of the orally administered dose in male rats was 61% (IBHK) and 46% in TMN alcohol. These values were a minimal estimation as potential biliary elimination was not included.

Details on distribution in tissues:

Disposition of the Administered 14C-IBHK and 14C-TMN alcohol in Tissues
Tissues from animals of both groups contained quantifiable radioactivity (Table 2). Tissues containing less than 0.005% of the administered dose, appear as 0.00% in the Tables.
Group 1. Single Oral Dose of 14C-IBHK
On average, a total of 0.8% of the 100 mg/ single oral dose of 14C-IBHK remained in the tissues 168 hours after dosing, most of which was found in the residual carcass and skin (0.26% and 0.30% of the administered dose, respectively).

Group 2. Single Oral Dose of 14C-TMN alcohol
On average, a total of 0.5% of the 100 mg/ single oral dose of 14C-TMN alcohol remained in the tissues 168 hours after dosing, most of which was found in the residual carcass and skin (0.17% and 0.21% of the administered dose, respectively).

Disposition of the Administered 14C-IBHK and 14C-TMN Alcohol in Tissues
Only the livers of the IBHK administered animals had an average of >1 μg-eq/g, all other tissues from the IBHK and TMN alcohol administered animals were <1 μg-eq/g. The thyroid of animal 08A7419 had a value of >8 μg-eq./g, while the other three animals had values that ranged from NQ(0.263) to 0.458 μg-eq./g values. The other tissues from animal 08A7419 were very consistent with the other animals in this dose group. It is believed the thyroid value for this animal was a result of contamination and not representative of the true value. Overall, the pattern of radioactive residues in
the terminal tissues was quite comparable between the two test materials.

Details on excretion:

Urinary Elimination of the Administered 14C-IBHK and 14C-TMN alcohol
Group 1. Single Oral Dose of 14C-IBHK
Following a single oral dose of 14C-IBHK (Group 1) a total of 57% of the administered dose was recovered in the urine/rinse (Table 2). IBHK-derived radioactivity was eliminated fairly quickly with 46 and 24% of total urinary elimination occurring during 0-12 and 12-24 hours post-dosing. (data not shown).
Group 2. Single Oral Dose of 14C-TMN alcohol
Following a single oral dose of 14C-TMN alcohol (Group 2) a total of 44% of the dose was recovered in the rat. Radioactivity was eliminated fairly quickly with 24, and 36% of total urinary elimination occurring during 0-12 and 12-24 hours post-dosing (data not shown).

Fecal Elimination of the Administered 14C-IBHK and 14C-TMN alcohol
At 168 hours post-dosing, 35 and 42% of the administered dose recovered in the respective feces (Table 2). The majority of the test-material derived radioactivity was eliminated in the 0-24 hour time interval.

Metabolite characterisation studies

Details on metabolites:

Metabolite(s) Profile and Quantitation
Both IBHK and TMN alcohol test materials were highly metabolized in male Crl:CD(SD) rats following a single 100 mg/kg bw oral dose with 60.5% and 44.0% of the IBHK and TMN radioactivity excreted in the urine, respectively.

IBHK and TMN alcohol Urines
A total of 27 metabolite peaks were detected in the urine from IBHK dosed rats. Three of the IBHK metabolite peaks were greater than 5% of the administered dose, U3 at 9.43%, U22 at 8.14% and U24 at 9.56%. A total of 16 metabolite peaks were detected in the urine from TMN alcohol administered rats. Two of the TMN metabolite peaks were greater than 5% of the administered dose, U23 at 13.5% and U27 at 10.8%. Seven “like” peaks (possessed the same HPLC-retention times) were observed between the IBHK and TMN urines. All of these “like” metabolites were <3.8% of the administered dose with most ~1% or less. The one exception was metabolite peak U27 which accounted for 10.8% of the administered dose in the TMN urine. However, the U27 metabolite peak only accounted for 0.43% of the administered dose in the IBHK urine. The HPLC analysis of the 0-12, 12-24 and 24-48 hr urine intervals (IBHK and TMN) showed consistent radiochemical profiles with no measurable change in metabolite distribution or new metabolites observed in these later time intervals (data not shown). No unchanged IBHK or TMN was observed in the respective urine samples.

IBHK and TMN alcohol Feces
Fecal samples from Crl:CD(SD) rats following a single 100 mg/kg bw oral dose of IBHK or TMN contained 34.8% and 41.6% of the excreted radioactivity, respectively. A total of 15 metabolite peaks were detected in the fecal extracts from IBHK dosed rats. Two of the IBHK metabolite peaks
were greater than 5% of the administered dose, F4 at 6.7%, and F20 at 8.2%. Fecal peak F20 was tentatively identified as parent IBHK by HPLC retention-time match with an authentic IBHK standard. A total of 7 metabolite peaks were detected in the fecal extracts from TMN dosed rats. Two of the TMN metabolite peaks were greater than 5% of the administered dose, F18 at 13.8% and F19 at 11.9%. These two fecal peaks (F18 & F19) were identified as parent TMN isomers by HPLC retention-time match with an authentic TMN standard. No “like” peaks (possessing the same retention times) were observed between the IBHK and TMN fecal extracts.

Urinary and Fecal Bioequivalence Comparison
Both test materials were highly metabolized, once absorbed, as no parent compound was seen in any of the urine samples analyzed. Of the 57% of the administered IBHK dose excreted in the urine through 168 hours post-dosing, 88% of the total was represented as unique metabolites to IBHK. Of the 44% of the administered TMN alcohol dose excreted in the urine through 168 hours post-dosing, 53% of the total was represented as unique metabolites to TMN. There were seven “like” metabolites, as determined via HPLC retention-time matches, in the IBHK and TMN urines. These data may suggest there might be some common metabolic pathways in these minor metabolites formation. However, the majority of the excreted metabolites for the two test materials is unique and are not shared.

There was 35-42% of the administered dose of IBHK and TMN excreted in the respective feces through 168 hours post-dosing. There were not “like” metabolites in these samples, based on HPLC retention-time matches.

The urinary and fecal metabolic data are consistent with IBHK and TMN sharing some minor metabolic pathways but the majority of the metabolism is unique.

Any other information on results incl. tables

Dose Suspension (Administration and Recovery of the Dose)

The measured concentration of total test material in each dose suspension was within 6% of the target concentration. The concentration of radioactivity in each dose suspension was within 20% of the target radioactivity.

The actual dose and radioactivity administered to rats via oral dosing was lower than the targeted dose 100 mg/kg and 500 μCi/kg, respectively. The lower-than-targeted values were consistent with less of the respective doses inadvertently administered to the animals (4 instead of 5 mL/kg). This resulted in the animals from both dose groups being administered 75 mg/kg; however, the nominal concentration of 100 mg/kg will be will be referred to in this report. The difference between the target and actual doses administered had no effects on the results of this study. There were no signs of toxicity observed in any animals following oral administration of ¹⁴C-IBHK or ¹⁴CTMN alcohol.

Total recovery of radioactivity from all the animals averaged 96 and 88% in the IBHK and TMN alcohol groups, respectively

Time-Course Concentration of Radioactivity and Pharmacokinetics in Plasma and RBC

Plasma

Plasma samples were analyzed for radioactivity for up to 168 hours post-dosing (Table 3). The kinetic parameters for the plasma concentration-time profiles of ¹⁴C-IBHK (Group 1) rats were derived using a two compartmental model and a one compartmental model for ¹⁴C-TMN alcohol (Group 2). Group 2 showed mono-phasic elimination, therefore no elimination t½β was calculated. The kinetic rates were derived from PK Solutions for Excel.

¹⁴C-IBHK Plasma Time -Course

The orally administered IBHK was readily absorbed and achieved the highest estimated plasma concentration (Cmax) at 3.5 hours (tmax) and ½ Cmax was reached within 12 hours (Table 4). The plasma elimination t½α was ~9 hours and the plasma elimination t½β was ~40 hours. The AUC of radioactivity at 100 mg/kg oral dose was 281 μg h g^-1.

¹⁴C-TMN Plasma Time-Course

The orally administered TMN alcohol was slowly absorbed and achieved the highest estimated concentration (C_max) at 14 hours (t_max) and ½ C_max was reached within 48 hours. Elimination of the radioactivity from plasma was mono-phasic with most of the elimination occurring during the (α) elimination phase. The plasma absorption t½ was ~3 hours. The plasma elimination t½ α was ~26 hours. The AUC of radioactivity at 100 mg/ oral dose was 194 μg h g^-1.

RBC

The RBC of Groups 1 and 2 were oxidized and the radioactivity determined by LSS (Table 3). The time-course ¹⁴C concentrations in RBC roughly paralleled the respective plasma time-course concentration profiles. Similar to the plasma, Group 1 RBC data were also best described by a two-compartment pharmacokinetic model. In Group 2, one male rat did not have sufficient data points to model; an n=3 was used to calculate the mean parameters. Group 2 showed mono-phasic elimination, therefore no elimination t½β was calculated. The calculated Cmax of radioactivity in RBC was approximately half (Groups 1 and 2) than the levels found in plasma. Elimination of the RBC-bound radioactivity (Group 1; IBHK) was similar to plasma with a rapid α (t½ ~ 12 hours) and a slower β phase (t½ = 121 hours). The α elimination of the RBC-bound radioactivity (Group 2; TMN alcohol) was slower than plasma (t½ ~ 46 hours).

14C-IBHK RBC Time-Course

The orally administered IBHK was readily absorbed and achieved the highest measured RBC concentration (C_max) at 3.75 hours (t_max) and ½ C_max was reached within 12 hours (Table 4). The plasma elimination t½α was ~12 hours. The RBC elimination t½β was ~120 hours. The AUC of radioactivity at 100 mg/ oral dose was 226 μg h g^-1.

¹⁴C-TMN RBC Time-Course

The orally administered TMN alcohol was slowly absorbed and achieved the highest measured RBC concentration (C_max) at 16 hours (t_max) and ½ C_max was reached within 48 hours. Elimination of the radioactivity from RBC was monophasic with the elimination occurring during the (α) elimination phase. The RBC absorption t½ was ~3 hours. The plasma elimination t½ α was ~46 hours. The AUC of radioactivity at 100 mg/ oral dose was 113 μg h g^-1.

Pharmacokinetic Bioequivalence Comparison in Plasma and RBC

The kinetic profile in plasma/RBC had some differences between test materials, primarily during the absorption phase. IBHK-derived radioactivity reached higher C_max concentrations (~3-4 times) at a shorter T_max (~4 times) than TMN (Table 4). Lower AUC values were found for the TMN alcohol (32-50%), consistent with the lower absorption seen (23%) for this test material. Terminal elimination rates were similar between the test materials, and both had shorter half-lives in plasma (26-40) than in RBC (46-121).

TABLE 2. Distribution of Radioactivity Recovered after Oral Administration of ¹⁴C-IBHK and ¹⁴C-TMN alcohol to CRL:CD(SD) Rats

			Mean + St. Dev.
Sample Class	Time (hr)	Sample	IBHK	TMN
Tissues		Blood	0.01 + 0.00	0.01 + 0.00
		Carcass	0.26 + 0.04	0.17 + 0.03
		GI Tract	0.13 + 0.02	0.11 + 0.03
		Kidneys	0.01 + 0.00	0.00 + 0.00
		Liver	0.08 + 0.02	0.04 + 0.01
		Skin	0.30 + 0.11	0.21 + 0.04
		Testes	0.00 + 0.00	0.00 + 0.00
		Thyroid	0.00 + 0.00	0.00 + 0.00
Tissues Total			0.79 + 0.14	0.54 + 0.05
Charcoal Trap	24	Ch. Trap	0.71 + 0.83	0.34 + 0.23
	48	Ch. Trap	--	--
	72	Ch. Trap	--	--
Charcoal Trap Total			0.71 + 0.83	0.34 + 0.23
CO2	12	CO2	0.83 + 0.06	0.33 + 0.17
	24	CO2	0.23 + 0.08	0.35 + 0.09
	48	CO2	--	--
	72	CO2	--	--
	96	CO2	--	--
CO2 Total			1.06 + 0.11	0.68 + 0.17
FCW		FCW	1.86 + 0.90	0.89 + 0.59
FCW Total			1.86 + 0.90	0.89 + 0.59
Feces	24	Feces	26.55 + 4.80	33.11 + 5.76
	48	Feces	6.35 + 1.88	6.35 + 1.30
	72	Feces	1.21 + 0.50	1.37 + 0.24
	96	Feces	0.38 + 0.12	0.46 + 0.08
	120	Feces	0.14 + 0.06	0.20 + 0.03
	144	Feces	0.10 + 0.09	0.06 + 0.03
	168	Feces	0.04 + 0.01	0.04 + 0.01
Feces Total			34.77 + 2.62	41.60 + 5.06
Urine/Rinse	12	Rinse Urine	5.80 + 1.32 20.41 + 7.52	3.07 + 1.95 7.46 + 1.09
	24	Rinse Urine	6.66 + 6.81 14.10 + 4.53	2.27 + 0.79 13.72 + 2.75
	48	Rinse Urine	0.99 + 0.51 8.98 + 1.73	0.90 + 0.49 10.56 + 0.78
	72	Rinse Urine	0.30 + 0.08 2.07 + 0.92	0.45 + 0.13 3.40 + 0.26
	96	Rinse Urine	0.13 + 0.04 0.55 + 0.22	0.23 + 0.08 1.09 + 0.18
	120	Rinse Urine	0.06 + 0.02 0.21 + 0.05	0.10 + 0.05 0.44 + 0.15
	144	Rinse Urine	0.03 + 0.01 0.11 + 0.03	0.04 + 0.03 0.21 + 0.05
	168	Rinse Urine	0.02 + 0.00 0.07 + 0.02	0.02 + 0.00 0.08 + 0.01
Urine/Rinse Total			56.95 + 5.67	44.04 + 5.55
Grand Total			96.14 + 5.74	88.08 + 2.59

Table 3  Time Course of Radioactivity in Plasma and RBC Following Oral Administration of ¹⁴C-IBHK and ¹⁴C-TMN alcohol to CRL:CD(SD) Rats

	IBHK		TMN
Time (hr)	Mean + St. Dev.		Mean + St. Dev.
0.08	0.096 + 0.076	NQ (0.119) + 0.006	0.096 + 0.047	NQ (0.083) + 0.006
0.17	0.795 + 0.282	0.328 + 0.146	0.284 + 0.066	NQ (0.105) + 0.014
0.25	1.258 + 0.262	0.541 + 0.105	0.449 + 0.088	0.108 + 0.012
0.5	2.061 + 0.519	0.959 + 0.229	0.762 + 0.239	0.163 + 0.055
1	5.195 + 1.525	2.384 + 0.657	1.301 + 0.595	0.329 + 0.133
2	10.831 + 4.047	5.325 + 2.006	2.189 + 0.985	0.477 + 0.178
3	14.590 + 2.903	7.312 + 1.342	3.149 + 0.525	0.783 + 0.164
6	10.712 + 2.968	5.256 + 1.334	2.949 + 1.240	0.442 + 0.070
12	6.006 + 1.054	3.600 + 0.464	3.439 + 0.738	1.908 + 1.383
24	4.589 + 1.412	2.998 + 0.906	4.218 + 1.887	2.448 + 1.498
48	1.042 + 0.077	1.003 + 0.096	1.348 + 0.113	0.656 + 0.064
72	0.445 + 0.056	0.924 + 0.309	0.669 + 0.045	0.495 + 0.053
96	0.285 + 0.037	0.649 + 0.104	0.300 + 0.046	0.263 + 0.027
120	0.199 + 0.018	0.609 + 0.097	0.195	0.298
144	0.135 + 0.019	0.496 + 0.098	0.130 + 0.044	0.212 + 0.060
168	0.100 + 0.013	0.491 + 0.083	0.088 + 0.017	0.386 + 0.382

TABLE 4. Kinetic Parameters of 14C -IBHK and 14C-TMN alcohol in the RBC of CRL:CD(SD Rats Following Oral Administration

	Mean + St. Dev.
PK Parameters	IBHK	TMN
tmax (h)	3.750 + 1.500	16.000 + 6.928
Cmax (ug/g)	7.600 + 0.906	2.767 + 1.629
Absorption t1/2 (h)	0.689 + 0.277	3.166 + 1.069
Elimination t1/2a (h)	12.431 + 3.871	46.349 + 8.473
Elimination t1/2B (h)	120.577 + 20.836	---
AUC0-t (ug h g^-1)	226.475 + 25.270	113.233 + 37.369
Clearance (ml kg^-1 h^-1)	---	---

Applicant's summary and conclusion

Conclusions:

These data show some similarities in the uptake, terminal elimination and extent of metabolism of IBHK and TMN in the male Crl:CD(SD) rat, with differences observed in the absorption rate and final metabolic profiles.

Executive summary:

In this study four male rats were administered either IBHK or TMN alcohol (TMN) at nominal concentrations of 100 mg test material/kg bw via single oral gavage. Plasma and RBC timecourses were conducted and excreta collected for 168 hours post-dosing. Selected tissues were collected at sacrifice and analyzed for radioactivity. Similarities and/or differences in pharmacokinetics and metabolism were evaluated via comparison of absorption, distribution, and excretion kinetics and metabolite profiles. These data were utilized to compare bioequivalence for these two compounds in the male Crl:CD(SD) rat.

Both test materials were appreciably absorbed, with a minimum of 61 and 46% of the administered dose for IBHK and TMN, respectively, recovered in urine, tissues (except GI tract), CO2 and final cage wash. There were no significant differences in tissue levels of respective test material derived radioactivity at 168 hours post-dosing.

The kinetic profile in plasma/RBC had some differences between test materials, primarily during the absorption phase. IBHK-derived radioactivity reached higher C_max concentrations (~3-4 fold) at a shorter T_max (~4 fold) than TMN. Lower AUC values were found for the TMN alcohol (32-50%), consistent with the lower absorption seen (23%) for this test material. Terminal elimination rates were similar between the test materials, and both had shorter halflives in plasma (26-40 hr) than in RBC (46-121 hr).

Both compounds were highly metabolized with the majority of their respective radioactivity excreted in the urine. Parent IBHK and TMN were not detected in the respective urine samples. A total of 27 and 16 metabolite peaks were detected in the urine from IBHK and TMN dosed rats. Three of the IBHK and two of the TMN metabolite peaks were greater than 5% of the administered dose. Seven “like” peaks (possessed the same HPLC-retention times) were observed between the IBHK and TMN urines. All of these “like” metabolites were <3.8% of the administered dose with most ~1% or less. The one exception was a TMN metabolite peak which accounted for ~10% of the administered dose in the urine. However, the corresponding IBHK urinary metabolite peak, with a comparable retention time, only accounted for < 0.5% of the administered dose. These data indicates the vast majority of the metabolites for IBHK and TMN are unique for each test material.

Overall, these data show some similarities in the uptake, terminal elimination and extent of metabolism of IBHK and TMN in the male Crl:CD(SD) rat, with differences observed in the absorption rate and final metabolic profiles.