Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 April 2015 to 15 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

extended postdose fasting in two animals with no impact on results or integrity of the study (see below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

yes

Remarks:

extended postdose fasting in two animals with no impact on results or integrity of the study (see below)

GLP compliance:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Appearance/physical state: Dark black solid
Storage conditions: Room temperature, protected from light, under inert gas

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMAL ACQUISITION, ASSIGNMENT TO STUDY AND MAINTENANCE
- The animal receipt, acclimation, randomization, and maintenance information is presented in Appendix C (attached).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

VEHICLE AND TEST ARTICLE PREPARATION
- Fresh vehicle, peanut oil (Arachis Oil, NF), was dispensed for use on study once and was stored at room temperature.
- The [14C] radiolabeled and non-radiolabeled test article were used as received from the Sponsor. No adjustment was made for purity. To prepare the dosing formulation, approximately 14.5 mg of radiolabeled and 314.3 mg of non-radiolabeled test article was combined with 55 mL of vehicle and mixed using a probe-type homogenizer to achieve a target concentration of 6 mg/mL (40.0 μCi/mL) at a total formulation weight of 51.0577 g. The dosing formulation was prepared on the day prior to dosing, sparged with nitrogen, and stored refrigerated at 2 to 8°C. Residual dosing formulation was stored refrigerated at 2 to 8 °C.

ANALYSIS OF DOSING FORMULATIONS
- Prior to dosing (Day -1) and after dosing (Day 1), single aliquots (approximately 0.09 g) of the dosing formulation were collected from the top, middle, and bottom portions of the container and, after appropriate dilution with dimethyl sulfoxide, were analyzed in duplicate by liquid scintillation counting (LSC) to determine the concentration of radioactivity and homogeneity of the dosing formulation. The dosing formulation was continuously stirred during sample collections.

CONCENTRATION ANALYSIS
- Prior to dosing (Day -1), triplicate aliquots (approximately 0.4550 g) of the dosing formulation were collected from the middle and sparged with nitrogen to determine the non-radiolabeled test article concentration. All analytical work was conducted by MPI Research, Inc., using an analytical method developed and validated under MPI Research, Inc., Study Number 1928-015.

RADIOCHEMICAL PURITY AND STABILITY ANALYSIS
- Prior to formulation of the test article, the radiochemical purity of neat samples of the stock radiolabeled test article was determined. These data are not reported but are maintained in the study file. Prior to dosing (Day -1) and after dosing (Day 1), duplicate aliquots (approximately 20 μL) of the dosing formulation were collected and, after appropriate dilution with water/acetonitrile 50/50 with 0.1% heptafluorobutyric acid, were analyzed by
high performance liquid chromatography (HPLC) for radiochemical purity and stability analysis. The dosing formulation was mixed by pipetting up and down during sample collections.

RESERVE SAMPLE AND TEST ARTICLE DISPOSITION
- A reserve sample from the lots of the radiolabeled and non-radiolabeled test article used on study was not collected because the duration of the study was less than 4 weeks.

TEST ARTICLE ADMINISTRATION
- The animals were fasted overnight prior to dosing, and food was returned approximately 2 to 2 hours and 43 minutes postdose.
- The dosing formulation was administered once via oral gavage. The actual amount administered was determined by weighing the dose syringe before and after dose administration. Individual doses were based on body weights collected on Day -1. Dosing formulations were continuously stirred during each loading of the dosing apparatus.
After the dose was administered, and as the gavage tube was removed, a dose wipe was collected into a plastic container, and was retained at room temperature prior to analysis by LSC to determine residual radioactivity.
- The radioactivity recovered was subtracted from the administered amount to give the actual radioactive dose administered. Results of dose site wipe radioanalysis are not reported but are maintained in the study file. Dose administration information is presented in the individual dose administration table (attached).

Duration and frequency of treatment / exposure:

Single dose

Doses / concentrations

Dose / conc.:

30 mg/kg bw/day

Remarks:

Target dose level was 30 mg/kg (200 µCi/kg) with a target dose volume of 5 mL/kg

No. of animals per sex per dose / concentration:

- Group 1: Four males (jugular vein cannulated)
- Group 2: Four females (jugular vein cannulated)
- Group 3: 4 males (bile duct cannulated)
- Group 4: Four males
- Group 5: Five males
- Group 6: Five females

Details on dosing and sampling:

RADIOANALYSIS SAMPLE COLLECTION – WHOLE BLOOD AND PLASMA
- Blood samples (approximately 0.4 mL) were collected from all Group 1 and 2 animals via the jugular vein cannula at 1, 2, 4, 8, 24, 48, and 72 hours postdose into tubes containing K2EDTA and placed on ice.
- Blood samples (approximately 0.5 mL) were collected from all Group 5 and 6 animals via the sublingual vein predose into tubes containing K2EDTA and placed on ice. Blood samples (maximum available) were collected from one Group 5 and 6 animal per time point via cardiac puncture after carbon dioxide inhalation at 2, 8, 24, 72, and 168 hours postdose into tubes containing K2EDTA and placed on ice.
- An aliquot (approximately 0.15 mL) of whole blood was collected from all collections (except the predose samples from Groups 5 and 6) and stored on wet ice until processed for radioanalysis.
- The remaining blood was centrifuged under refrigeration to obtain plasma and the samples were stored on wet ice until processed for radioanalysis.

RADIOANALYSIS SAMPLE COLLECTION – EXCRETA
- Urine and feces (if available) were collected from all Group 1 and 2 animals predose and at 0 to 8, 8 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144, and 144 to 168 hours postdose. Each sample was collected into a plastic container on wet ice and maintained under this condition until processed for radioanalysis.

RADIOANALYSIS SAMPLE COLLECTION – BILE
- Bile was collected from Group 3 animals predose and at 0 to 8, 8 to 24, 24 to 48, and 48 to 72 hours postdose.
- Each sample was collected into a plastic container on wet ice and maintained under this condition until processed for radioanalysis.

RADIOANALYSIS SAMPLE COLLECTION – CAGE RESIDUE
- The cages were rinsed with deionized water following each excreta collection at 24, 48, 72, 96, 120, and 144 hours postdose.
- Following the final excreta collection at 168 hours postdose, the cages were rinsed with 1% trisodium phosphate solution and wiped using gauze pads.
- Each cage rinse and wipe sample was collected into a separate plastic container at room temperature and maintained under this condition until processed for radioanalysis.

RADIOANALYSIS SAMPLE COLLECTION – EXPIRED AIR TRAPS
- Expired organic volatiles and expired radiolabeled carbon dioxide (14CO2) were collected from Group 4 animals by drawing air through the metabolism cage enclosed in airtight housing, and trapping. Expired organic volatiles were passed through a trap containing activated charcoal and through a series of at least two bubbler flasks containing a solution of 2N sodium hydroxide.
- The expired air trap samples (CO2, volatiles) were collected at approximately 0 to 24, 24 to 48, and 48 to 72 hours postdose. The sodium hydroxide solution was collected into a plastic container at room temperature and maintained under this condition until radioanalysis. The trap contents were immediately replaced with fresh activated charcoal or solution, as appropriate.

RADIOANALYSIS SAMPLE COLLECTION – CARCASS
- At the terminal time point of 168 hours postdose, Group 1 and 2 animals were euthanized by carbon dioxide inhalation followed by cervical dislocation. The carcass was collected from each animal and processed for radioanalysis.

QUANTITATIVE WHOLE BODY AUTORADIOGRAPHY (QWBA) – FREEZING OF CARCASSES
- At each terminal time point of 2, 8, 24, 72, and 168 hours postdose, one Group 5 and 6 animal per time point were deeply anesthetized with carbon dioxide and were immersed in a hexane/dry ice bath for 11 to 35 minutes. Following immersion in the hexane/dry ice bath, each carcass was drained, blotted dry, and was placed in an appropriately labeled plastic bag, and was stored frozen at -10 to -30°C until QWBA analysis.

QUANTITATIVE WHOLE BODY AUTORADIOGRAPHY (QWBA) – EMBEDDING OF CARCASSES
- Prior to embedding, the animal carcasses were removed from frozen storage. The tail was removed as close to the rectal tissues as possible without removing any sex organs. The ears were amputated at the base, the forelimbs at mid forearm, and the hindlimbs above the ankle joint. The carcasses were positioned in a supine position in an appropriately sized embedding frame, covered with 5% carboxymethylcellulose (CMC), and allowed to freeze until solid at -10 to -30°C.

QUANTITATIVE WHOLE BODY AUTORADIOGRAPHY (QWBA) – SECTIONING OF CARCASSES
- At least ten quality control standards were placed into the frozen blocks prior to sectioning and were used for section thickness quality control. Sections approximately 30 to 50 μm thick were taken from the sagittal plane and captured on adhesive tape.
- Appropriate sections selected at various levels of interest in the block were collected to encompass the required tissues and biological fluids, where possible.

QUANTITATIVE WHOLE BODY AUTORADIOGRAPHY (QWBA) – MOUNTING OF SECTIONS
- The sections were allowed to dry by sublimation at -10 to -30°C for at least 1 hour. The appropriate number of sections was mounted on a mat board, and exposed along with 14C-spiked calibration standards to 14C-sensitive imaging plates.

QUANTITATIVE WHOLE BODY AUTORADIOGRAPHY (QWBA) – EXPOSURE AND SCANNING OF SECTIONS
- The imaging plates and sections were enclosed in exposure cassettes and allowed to expose for at least four days in a steel-lined lead safe. The sections were removed from the imaging plate at the end of the exposure time, and the plates were scanned to produce images.

QUANTITATIVE WHOLE BODY AUTORADIOGRAPHY (QWBA) – IMAGE ANALYSIS
- Quantification, relative to the calibration standards, was performed by image densitometry. A standard curve was constructed from the integrated response (MDC/mm2) and the nominal concentrations of the 14C-calibration standards. Relative error was less than 0.5% for each point on the curve. A lower limit of quantification (LLOQ) was applied to the data. The LLOQ was determined to be 382 ng-eq/g by using the radioactive concentration of the lowest calibration standard used to generate a calibration curve divided by the specific activity of the dose formulation (μCi/μg). Artifacts were excluded from analysis during image processing.

STUDY TERMINATION
- Following excreta collection at 72 hours postdose, Group 3 and 4 animals were euthanized by carbon dioxide inhalation followed by cervical dislocation. The carcasses were stored at -10 to -30°C pending disposal.

SAMPLE PREPARATION AND ANALYSIS
- Aliquots of dosing formulation, plasma, urine, bile, cage rinse, expired air, carcass, after homogenization, and dose and cage wipe extracts were mixed well and sampled in duplicate or triplicate for direct analysis of radioactivity. Whole blood and feces homogenates, after homogenization, were aliquoted in triplicate for analysis of radioactivity via LSC by oxidation.
- All radioactive samples collected were counted by LSC for at least 5 minutes or 100,000 counts in duplicate or triplicate, sample size allowing. If the results for the sample duplicate or triplicate (calculated at dpm/g sample) differed by more than 15% from the mean value, a new sample was re-aliquoted, or re-oxidized, as appropriate, and reanalyzed, sample size permitting. This specification was met for all sample aliquots that had radioactivity
greater than 200 dpm. Specific radioanalysis information and sample storage conditions following radioanalysis are presented in the radioanalysis information table (attached).

METABOLITE PROFILING AND IDENTIFICATION
- Plasma, urine, fecal extracts, and bile were analyzed for metabolite profiling and identification as presented in Appendix M (attached).

Statistics:

- Statistical analysis was not performed. However, descriptive statistics, including means and standard deviations, were calculated.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

See discussion below

Main ADME results 2

Type:

distribution

Results:

See discussion below

Main ADME results 3

Type:

metabolism

Results:

See discussion below

Main ADME results 4

Type:

excretion

Results:

See discussion below

Toxicokinetic / pharmacokinetic studies

Details on absorption:

ABSORPTION
- For Group 1 male rats orally dosed with [14C] test item, the peak of radioactivity or Tmax in plasma was observed at 2 hours postdose. The mean peak concentration in plasma or Cmax at 2 hours was 1,644.89 ± 460.50 ng-eq/g. The Cmax was followed by a slight decline through 8 hours postdose (Distribution Phase), and a steeper decline thereafter (Elimination Phase). The last measurable mean plasma value at 48 hours postdose was 22.91 ± 45.81 ng-eq/g. The plasma value at 72 hours postdose did not have measurable levels of test article, and was recorded as zero.
- For Group 2 female rats orally dosed with [14C] test item, the peak of radioactivity or Tmax in plasma was observed at 1 hour postdose. The mean peak concentration in plasma or Cmax at 1 hour was 1,850.39 ± 431.95 ng-eq/g. The Cmax was followed by a steady decline through 72 hours postdose, and distinct Distribution and Elimination phases were indistinguishable. The last measurable mean plasma value at 72 hours postdose was 38.67 ± 77.34 ng-eq/g.
- For Group 5 male rats orally dosed with [14C] test item, terminal blood was removed and analyzed following euthanasia at all QWBA timepoints. The plasma values for this group were single point analyses, and statistical analysis was not possible. Plasma concentration values in this group correlated well with the plasma concentration values obtained for Group 1 orally dosed male rats.
- For Group 6 female rats orally dosed with [14C] test item, terminal blood was removed and analyzed following euthanasia at all QWBA timepoints. The plasma values for this group were single point analyses, and statistical analysis was not possible. Plasma concentration values in this group correlated well with the plasma concentration values obtained for Group 2 orally dosed female rats.
- Blood to plasma ratios were calculated for all animals in all groups where blood was sampled. For Group 1 males, blood to plasma ratios ranged from a mean average of 0.62 to 0.86 through 24 hours postdose. These values are considered normal, indicative of low partitioning to the erythrocytes. At 48 hours postdose, the ratio could only be calculated for one animal (104M), which was 1.95 – indicative of higher partitioning to the erythrocytes. At 72 hours postdose, the blood to plasma ration could not be calculated, as the plasma values were recorded as zero. For Group 2 females, mean blood to plasma ratios ranged from 0.68 to 0.87 through 8 hours postdose, after which mean values rose to 1.57 at 24 hours postdose, and 5.48 at 48 hours postdose, indicative of high partitioning to erythrocytes with increased time. Blood to plasma ratios for both male and females for both Groups 5 and 6 (QWBA animals) followed the same pattern outlined for Groups 1 and 2 males and females.

Details on distribution in tissues:

DISTRIBUTION
- Following oral administration of [14C] test item to intact male and female rats, the test substance-derived radioactivity was widely distributed in both sexes. In general, distribution was faster in females, as evidenced by a Cmax of 2 hours for females versus 8 hours for males. Additionally, the quantitative levels in certain common tissues were higher for females than males.
- The limit of quantitation was determined to be approximately 382 nanogram-equivalents of [14C] test item per gram of tissue. A concentration of 2,000 ng-eq/g was logically selected as a threshold value representing significant exposure to the tissues analysed (based on the results obtained). At this selected threshold value, 16 of 34 tissues quantitated were noted as above threshold at the observed tissue Cmax of 8 hours postdose in males, and 18 of 34 tissues quantitated were noted as above threshold at the observed tissue Cmax of 2 hours postdose in females. As would be expected for an orally administered test article, the majority of the tissues noted as positive are part of the gastrointestinal tract (and associated organ systems), for both males and females.
- For intact male rats, the 16 tissues noted with concentrations greater than the selected threshold value of 2,000 ng-eq/g at 8 hours included (listed from highest to lowest concentration): Stomach contents (2,071,270 ng-eq/g); Cecum contents (1,020,869 ng-eq/g); Large intestinal contents (33,968 ng-eq/g); Liver (14,449 ng-eq/g); Adrenal gland (9,686 ng-eq/g); Kidney (5,026 ng-eq/g); Fat, brown (3,854 ng-eq/g); Pancreas (3,838 ng-eq/g); Thyroid gland (3,229 ng-eq/g); Cecum (2,982 ng-eq/g); Salivary gland (2,799 ng-eq/g); Lung (2,680 ng-eq/g); Myocardium (2,647 ng-eq/g); Skin (2,560 ng-eq/g); Spleen (2,431 ng-eq/g); and Large intestine (2,008 ng-eq/g). Several tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 2 hours postdose (the first QWBA timepoint) included (listed from highest to lowest concentration): Small intestinal contents (882,343 ng-eq/g); Small intestine (18,998 ng-eq/g); Urine (12,447 ng-eq/g); Urinary bladder (4,993 ng-eq/g); and Stomach (2,733 ng-eq/g).
- Other tissues in the males where distribution was noted as positive, but that was less than the selected threshold included: Bone (647 ng-eq/g only observed at 168 hours postdose); Cerebellum (705 ng-eq/g only observed at 2 hours postdose); Medulla (670 ng-eq/g only observed at 2 hours postdose); Bone marrow (Cmax of 1,660 ng-eq/g at 8 hours postdose); Prostate gland (Cmax of 1,600 ng-eq/g at 8 hours postdose); Thymus (Cmax of 1,551 ng-eq/g at 8 hours postdose); Muscle (Cmax of 1,251 ng-eq/g at 8 hours postdose); Blood (Cmax of 1,087 ng-eq/g at 8 hours postdose); Testes (Cmax of 1,084 ng-eq/g at 8 hours postdose); Fat, white (Cmax of 688 ng-eq/g at 2 hours postdose); Olfactory lobe (Cmax of 501 ng-eq/g at 2 hours postdose); Cerebrum (Cmax of 681 ng-eq/g at 2 hours postdose); and Eye, lens (382 ng-eq/g only observed at 8 hours postdose).
- For intact female rats, the 18 tissues noted with concentrations greater than the threshold value of 2,000 ng-eq/g at 2 hours included (listed from highest to lowest concentration): Stomach contents (1,261,640 ng-eq/g); Small intestinal contents (1,077,609 ng-eq/g); Small intestine (29,410 ng-eq/g); Liver (17,158 ng-eq/g); Stomach (17,068 ng-eq/g); Adrenal gland (14,986 ng-eq/g); Kidney (6,590 ng-eq/g); Salivary gland (6,329 ng-eq/g); Thyroid gland (5,492 ng-eq/g); Fat, brown (5,384 ng-eq/g); Pancreas (5,110 ng-eq/g); Lung (4,281 ng-eq/g); Spleen (3,194 ng-eq/g); Myocardium (3,143 ng-eq/g); Uterus (2,746 ng-eq/g); Olfactory lobe (2,135 ng-eq/g); Thymus (2,135 ng-eq/g); and Bone marrow (2,068 ng-eq/g). Several tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 8 hours postdose (the second QWBA timepoint) included (listed from highest to lowest concentration): Large intestinal contents (1,447,956 ng-eq/g); Cecum contents (335,591 ng-eq/g); Cecum (12,183 ng-eq/g); Eye, lens (2,855 ng-eq/g); and Large intestine (2,736 ng-eq/g). Two tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 24 hours postdose (the third QWBA timepoint) included (listed from highest to lowest concentration): Skin (4,577 ng-eq/g); and Ovary (3,332 ng-eq/g).
- Other tissues in the females where distribution was noted that was less than threshold included: Bone (1,579 ng-eq/g only observed at 168 hours postdose); Cerebellum (1,489 ng-eq/g Cmax observed at 2 hours postdose); Medulla (1,399 ng-eq/g Cmax observed at 2 hours postdose); Muscle (Cmax of 1,408 ng-eq/g at 2 hours postdose); Blood (Cmax of 1,315 ng-eq/g at 2 hours postdose); Fat, white (Cmax of 1,625 ng-eq/g at 2 hours postdose); and Cerebrum (Cmax of 1,530 ng-eq/g at 2 hours postdose).

Details on excretion:

ELIMINATION
- For Group 1 orally dosed male rats, the main route of elimination of [14C] test item was fecal excretion. The mean fecal recovery of the radioactive dose through 168 hours postdose was 71.93% ± 2.82%. Zero percent of the total dose recovered from the feces was recovered within the first 8 hours postdose, and approximately 75.03% of the total dose recovered from the feces was recovered from 8-24 hours postdose. A total of 97.37% of the total dose recovered from the feces was recovered within the first 48 hours postdose.
- Urinary excretion of [14C] test item was relatively low in male animals, with a total recovery of just 4.25% ± 0.70% of the total dose recovered through 168 hours postdose.
- Mean total recovery of [14C] test item from carcasses at 168 hours postdose was 0.87% ± 0.19% for orally dosed male rats.
- For Group 1 male rats, mean total recovery of the orally administered radioactive dose of [14C] test item from all sources through 168 hours postdose was 78.02% ± 2.56%, with individual total recovery values of: 76.88%, 77.86%, 75.71%, and 81.62%.
- The radioactive material unaccounted for represents approximately 21.98% of the total orally administered dose of [14C] test item to Group 1 male rats.
- For Group 2 orally dosed female rats, the main route of elimination of [14C] test item was also fecal excretion. The mean fecal recovery of the radioactive dose through 168 hours postdose was 76.00% ± 10.43%. Just 0.30% of the total dose recovered from the feces was recovered within the first 8 hours postdose, and approximately 55.46% of the total dose recovered from the feces was recovered from 8-24 hours postdose. A total of 85.49% of the total dose recovered from the feces was recovered within the first 48 hours postdose. The mean percentage of the radioactive dose collected in the feces of females was slightly greater than males (76.00% versus71.93%), and the rate of fecal elimination was slower than the rate observed in males.
- Urinary excretion of [14C] test item was relatively low in female animals, with a total recovery of just 4.85% ± 0.20% of the total dose recovered through 168 hours postdose. The mean percentage recovery in urine for females compares well with that for males (4.85% versus 4.25%).
- Mean total recovery of [14C]test item from carcasses at 168 hours postdose was 1.62% ± 0.62% – nearly double that observed for males (1.62% versus 0.87%).
- For Group 2 female rats, mean total recovery of the orally administered radioactive dose of [14C] test item from all sources through 168 hours postdose was 83.70% ± 9.75%, with individual total recovery values of: 82.64%, 72.94%, 96.63%, and 82.60%. The mean total recovery was better for females (83.70% versus 71.93%).
- The radioactive material unaccounted for represents approximately 16.30% of the total orally administered dose of [14C] test item, which was slightly better than the value observed for males (21.98%).
- Biliary excretion of [14C] test item in Group 3 male rats was significant, with a cumulative mean recovery of 31.06% ± 2.44% of the total dose administered recovered through 72 hours postdose. Recovery of the bile in individual animals was consistent, ranging from a low of 27.55% to a high of 32.91%. The majority of the radioactivity in bile-duct canulated male animals was observed in the bile from 0-8 hours, with approximately 51.19% of the total dose recovered from the bile obtained in the first 8 hours, and 92.56% of the total dose collected from the bile recovered within the first 24 hours postdose.
- Excretion of [14C] test item in expired air in Group 4 male rats over 72 hours postdose was zero percent via this excretion route.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

METABOLITE PROFILING AND IDENTIFICATION
- Results of the metabolite profiling and identification are presented in Appendix M (attached).
- The [14C] test item starting material is a mixture of components which is extensively metabolized, with little to none of the starting material observed in any of the matrices examined in this study.
- The major metabolite noted in feces of males, greater than five percent of dose, was M39, which was 23.8% of dose at 8-24 hours, and 11.6% of dose at 24-48 hours postdose. In females, the M39 was 14.5% of dose at 24-48 hours, 12.9% of dose at 24-48 hours, and 5.4% of dose at 48-72 hours postdose.
- The major metabolite noted in bile of males, greater than five percent of dose, was also M39, which was 4.4% of dose at 0-8 hours, 8.4% at 8-24 hours, and 2.0% of dose at 24-48 hours postdose. Bile collection from bile-duct canulated female rats was not part of the study design.
- Metabolite M39 was not observed in urine from males, but was observed in urine from females at 0.2% of dose, only in the 8-24 hour postdose sample. It should also be noted that all the metabolites characterized were less than one percent of dose in urine, which is not the major route of excretion for this test material.
- Metabolite M39 was also observed in plasma from 2, 8, and 24 hours postdose pooled plasma at 62.1% ROI in males and 68.6% ROI in females. It should be noted that with plasma, only a percent region of interest (% ROI) can be calculated, and not a percent of dose.

METABOLISM
- The [14C] test item starting material is a mixture of components which is extensively metabolized, with little to none of the starting material observed in any of the matrices examined in this study.
- The major metabolite noted in feces of males, greater than five percent of dose, was M39, which was 23.8% of dose at 8-24 hours, and 11.6% of dose at 24-48 hours postdose. In females, the M39 was 14.5% of dose at 24-48 hours, 12.9% of dose at 24-48 hours, and 5.4% of dose at 48-72 hours postdose.
- The major metabolite noted in bile of males, greater than five percent of dose, was also M39, which was 4.4% of dose at 0-8 hours, 8.4% at 8-24 hours, and 2.0% of dose at 24- 48 hours postdose. Bile collection from bile-duct canulated female rats was not part of the study design.
- Metabolite M39 was not observed in urine from males, but was observed in urine from females at 0.2% of dose, only in the 8-24 hour postdose sample. It should also be noted that all the metabolites characterized were less than one percent of dose in urine, which is not the major route of excretion for this test material.
- Metabolite M39 was also observed in plasma from 2, 8, and 24 hours postdose pooled plasma at 62.1% ROI in males and 68.6% ROI in females. It should be noted that with plasma, only a percent region of interest (% ROI) can be calculated, and not a percent of dose.

Any other information on results incl. tables

RADIOACTIVITY ANALYSIS

- Results of the radioactivity concentration and homogeneity analysis are presented in Section 1 of Appendix B, attached).

- The average radiochemical concentration of the formulation from the predose and postdose measurements was 86753398 dpm/g or 39.1 μCi/g. The predose and postdose concentrations did not differ markedly as the absolute percent difference was -11.34%. The low relative standard deviations predose and postdose, 5.56% and 5.24%, respectively, and the small change in the mean radiochemical concentration predose and postdose, show that the radioactivity in this formulation was homogeneous throughout the dosing period.

- The final formulation parameters used to determine the dose administration and concentration of radioactivity were based on the mean of the predose and postdose values and are presented below.

- Radioactivity Concentration of Formulation: 86753398 dpm/g (39.1 μCi/g)

- Test Substance Concentration in Formulation: 6.45183 mg/g

- Specific Activity of Formulation: 6.0569 μCi/mg

 

CONCENTRATION ANALYSIS

- Results of the non-radiolabeled test article concentration analysis are presented in Section 2 of Appendix B (attached).

- The average chemical concentration of the formulation from the predose and postdose analysis was 5.7578 mg/mL. The percent relative standard deviation between the predose and postdose aliquots was 1.070%. The concentration value was calculated based on the dpm/g and dose concentration (mg/mL) determined through HPLC analysis.

 

RADIOCHEMICAL COMPOSITION, PURITY AND STABILITY ANALYSIS

- Results of the radiochemical purity and stability analysis are presented in Section 3 of Appendix B (attached).

- The test article is a known complex mixture, used in industrial lubricants. The main components of interest are a dimer and monomer (see attached table). The Certificate of Analysis for the radiolabeled dimer/monomer mixture received from PerkinElmer (PerkinElmer Custom Services, Boston, MA) for the Lubrizol 107439348, dimer/monomer mixture, [monosubstituted aminophenyl-14C(U)] indicated a dimer/monomer ratio of 70.24%:13.18% (C of A shown in Section 2 of Appendix B, attached).

- Radiochemical composition analysis, with a similar method developed in-house indicated a dimer/monomer ratio of 69.47%:12.21% (results are not reported but are maintained in the study data).

 

WHOLE BLOOD AND PLASMA

- Mean whole blood and plasma concentration values are illustrated in Figure 1 (attached) and summarized in Table 1 (attached).

-For Group 1 male rats orally dosed with [14C] test item, the peak of radioactivity or Tmax in plasma was observed at 2 hours postdose. The mean peak concentration in plasma or Cmax at 2 hours was 1,644.89 ± 460.50 ng-eq/g. The Cmax was followed by a slight decline through 8 hours postdose (Distribution Phase), and a steeper decline thereafter (Elimination Phase). The last measurable mean plasma value at 48 hours postdose was 22.91 ± 45.81 ng-eq/g. The plasma value at 72 hours postdose did not have measurable levels of test article, and was recorded as zero.

- For Group 2 female rats orally dosed with [14C] test item, the peak of radioactivity or Tmax in plasma was observed at 1 hour postdose. The mean peak concentration in plasma or Cmax at 1 hour was 1,850.39 ± 431.95 ng eq/g. The Cmax was followed by a steady decline through 72 hours postdose, and distinct Distribution and Elimination phases were indistinguishable. The last measurable mean plasma value at 72 hours postdose was 38.67 ± 77.34 ng-eq/g.

-For Group 5 male rats orally dosed with [14C] test item, terminal blood was removed and analyzed following euthanasia at all QWBA timepoints. The plasma values for this group were single point analyses, and statistical analysis was not possible. Plasma concentration values in this group correlated well with the plasma concentration values obtained for Group 1 orally dosed male rats.

- For Group 6 female rats orally dosed with [14C] test item, terminal blood was removed and analyzed following euthanasia at all QWBA timepoints. The plasma values for this group were single point analyses, and statistical analysis was not possible. Plasma concentration values in this group correlated well with the plasma concentration values obtained for Group 2 orally dosed female rats.

- Blood to plasma ratios were calculated for all animals in all groups where blood was sampled. For Group 1 males, blood to plasma ratios ranged from a mean average of 0.62 to 0.86 through 24 hours postdose. These values are considered normal, indicative of low partitioning to the erythrocytes. At 48 hours postdose, the ratio could only be calculated for one animal (104M), which was 1.95 – indicative of higher partitioning to the erythrocytes. At 72 hours postdose, the blood to plasma ration could not be calculated, as the plasma values were recorded as zero. For Group 2 females, mean blood to plasma ratios ranged from 0.68 to 0.87 through 8 hours postdose, after which mean values rose to 1.57 at 24 hours postdose, and 5.48 at 48 hours postdose, indicative of high partitioning to erythrocytes with increased time. Blood to plasma ratios for both male and females for both Groups 5 and 6 (QWBA animals) followed the same pattern outlined for Groups 1 and 2 males and females.

 

RECOVERY OF RADIOACTIVITY

- Mean cumulative percent recoveries of urine, feces, bile, and cage rinse are illustrated in Figure 2 (attached), and percent recoveries (including cage wipe and carcasses) are summarized in Table 3 (attached).

-For Group 1 orally dosed male rats, the main route of elimination of [14C] test item was fecal excretion. The mean fecal recovery of the radioactive dose through 168 hours postdose was 71.93% ± 2.82%. Zero percent of the total dose recovered from the feces was recovered within the first 8 hours postdose, and approximately 75.03% of the total dose recovered from the feces was recovered from 8-24 hours postdose. A total of 97.37% of the total dose recovered from the feces was recovered within the first 48 hours postdose.

- Urinary excretion of [14C] test item was relatively low in male animals, with a total recovery of just 4.25% ± 0.70% of the total dose recovered through 168 hours postdose.

- Mean total recovery of [14C] test item from carcasses at 168 hours postdose was 0.87% ± 0.19% for orally dosed male rats. For Group 1 male rats, mean total recovery of the orally administered radioactive dose of [14C] test item from all sources through 168 hours postdose was 78.02% ± 2.56%, with individual total recovery values of: 76.88%, 77.86%, 75.71%, and 81.62%.

- The radioactive material unaccounted for represents approximately 21.98% of the total orally administered dose of [14C] test item to Group 1 male rats.

- For Group 2 orally dosed female rats, the main route of elimination of [14C] test item was also fecal excretion. The mean fecal recovery of the radioactive dose through 168 hours postdose was 76.00% ± 10.43%. Just 0.30% of the total dose recovered from the feces was recovered within the first 8 hours postdose, and approximately 55.46% of the total dose recovered from the feces was recovered from 8-24 hours postdose. A total of 85.49% of the total dose recovered from the feces was recovered within the first 48 hours postdose. The mean percentage of the radioactive dose collected in the feces of females was slightly greater than males (76.00% versus71.93%), and the rate of fecal elimination was slower than the rate observed in males.

- Urinary excretion of [14C] test item was relatively low in female animals, with a total recovery of just 4.85% ± 0.20% of the total dose recovered through 168 hours postdose. The mean percentage recovery in urine for females compares well with that for males (4.85% versus 4.25%).

- Mean total recovery of [14C] test item from carcasses at 168 hours postdose was 1.62% ± 0.62% – nearly double that observed for males (1.62% versus 0.87%).

- For Group 2 female rats, mean total recovery of the orally administered radioactive dose of [14C] test item from all sources through 168 hours postdose was 83.70% ± 9.75%, with individual total recovery values of: 82.64%, 72.94%, 96.63%, and 82.60%. The mean total recovery was better for females (83.70% versus 71.93%).

- The radioactive material unaccounted for represents approximately 16.30% of the total orally administered dose of [14C] test item, which was slightly better than the value observed for males (21.98%).

- Biliary excretion of [14C] test item in Group 3 male rats was significant, with a cumulative mean recovery of 31.06% ± 2.44% of the total dose administered recovered through 72 hours postdose. Recovery of the bile in individual animals was consistent, ranging from a low of 27.55% to a high of 32.91%. The majority of the radioactivity in bileduct canulated male animals was observed in the bile from 0-8 hours, with approximately 51.19% of the total dose recovered from the bile obtained in the first 8 hours, and 92.56% of the total dose collected from the bile recovered within the first 24 hours postdose.

- Excretion of [14C] test item in expired air in Group 4 male rats over 72 hours postdose was zero percent via this excretion route.

 

QUANTITATIVE WHOLE BODY AUTORADIOGRAPHY

- Following oral administration of [14C] test item to intact male and female rats, the test substance-derived radioactivity was widely distributed in both sexes. In general, distribution was faster in females, as evidenced by a Cmax of 2 hours for females versus 8 hours for males. Additionally, the quantitative levels in certain common tissues were higher for females than males. The limit of quantitation was determined to be approximately 382 nanogram-equivalents of [14C] test item per gram of tissue. A concentration of 2,000 ng-eq/g was logically selected as a threshold value representing significant exposure to the tissues analyzed (based on the results obtained). At this selected threshold value, 16 of 34 tissues quantitated were noted as above threshold at the observed tissue Cmax of 8 hours postdose in males, and 18 of 34 tissues quantitated were noted as above threshold at the observed tissue Cmax of 2 hours postdose in females. As would be expected for an orally administered test article, the majority of the tissues noted as positive are part of the gastrointestinal tract (and associated organ systems), for both males and females.

- For intact male rats, the 16 tissues noted with concentrations greater than the selected threshold value of 2,000 ng-eq/g at 8 hours included (listed from highest to lowest concentration): Stomach contents (2,071,270 ng-eq/g); Cecum contents (1,020,869 ng eq/g); Large intestinal contents (33,968 ng-eq/g); Liver (14,449 ng-eq/g); Adrenal gland (9,686 ng-eq/g); Kidney (5,026 ng-eq/g); Fat, brown (3,854 ng-eq/g); Pancreas (3,838 ng-eq/g); Thyroid gland (3,229 ng-eq/g); Cecum (2,982 ng-eq/g); Salivary gland (2,799 ng-eq/g); Lung (2,680 ng-eq/g); Myocardium (2,647 ng-eq/g); Skin (2,560 ng-eq/g); Spleen (2,431 ng-eq/g); and Large intestine (2,008 ng-eq/g). Several tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 2 hours postdose (the first QWBA timepoint) included (listed from highest to lowest concentration): Small intestinal contents (882,343 ng-eq/g); Small intestine (18,998 ng-eq/g); Urine (12,447 ng-eq/g); Urinary bladder (4,993 ng-eq/g); and Stomach (2,733 ng-eq/g).

- Other tissues in the males where distribution was noted as positive, but that was less than the selected threshold included: Bone (647 ng-eq/g only observed at 168 hours postdose); Cerebellum (705 ng-eq/g only observed at 2 hours postdose); Medulla (670 ng-eq/g only observed at 2 hours postdose); Bone marrow (Cmax of 1,660 ng-eq/g at 8 hours postdose); Prostate gland (Cmax of 1,600 ng-eq/g at 8 hours postdose); Thymus (Cmax of 1,551 ng eq/g at 8 hours postdose); Muscle (Cmax of 1,251 ng-eq/g at 8 hours postdose); Blood (Cmax of 1,087 ng-eq/g at 8 hours postdose); Testes (Cmax of 1,084 ng eq/g at 8 hours postdose); Fat, white (Cmax of 688 ng-eq/g at 2 hours postdose); Olfactory lobe (Cmax of 501 ng-eq/g at 2 hours postdose); Cerebrum (Cmax of 681 ng-eq/g at 2 hours postdose); and Eye, lens (382 ng-eq/g only observed at 8 hours postdose).

- For intact female rats, the 18 tissues noted with concentrations greater than the threshold value of 2,000 ng-eq/g at 2 hours included (listed from highest to lowest concentration): Stomach contents (1,261,640 ng-eq/g); Small intestinal contents (1,077,609 ng-eq/g); Small intestine (29,410 ng-eq/g); Liver (17,158 ng-eq/g); Stomach (17,068 ng-eq/g); Adrenal gland (14,986 ng-eq/g); Kidney (6,590 ng-eq/g); Salivary gland (6,329 ng-eq/g); Thyroid gland (5,492 ng-eq/g); Fat, brown (5,384 ng-eq/g); Pancreas (5,110 ng-eq/g); Lung (4,281 ng-eq/g); Spleen (3,194 ng-eq/g); Myocardium (3,143 ng-eq/g); Uterus (2,746 ng-eq/g); Olfactory lobe (2,135 ng-eq/g); Thymus (2,135 ng-eq/g); and Bone marrow (2,068 ng-eq/g). Several tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 8 hours postdose (the second QWBA timepoint) included (listed from highest to lowest concentration): Large intestinal contents (1,447,956 ng-eq/g); Cecum contents (335,591 ng-eq/g); Cecum (12,183 ng eq/g); Eye, lens (2,855 ng-eq/g); and Large intestine (2,736 ng-eq/g). Two tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 24 hours postdose (the third QWBA timepoint) included (listed from highest to lowest concentration): Skin (4,577 ng-eq/g); and Ovary (3,332 ng-eq/g).

- Other tissues in the females where distribution was noted that was less than threshold included: Bone (1,579 ng-eq/g only observed at 168 hours postdose); Cerebellum (1,489 ng-eq/g Cmax observed at 2 hours postdose); Medulla (1,399 ng-eq/g Cmax observed at 2 hours postdose); Muscle (Cmax of 1,408 ng-eq/g at 2 hours postdose); Blood (Cmax of 1,315 ng-eq/g at 2 hours postdose); Fat, white (Cmax of 1,625 ng-eq/g at 2 hours postdose); and Cerebrum (Cmax of 1,530 ng-eq/g at 2 hours postdose).

Applicant's summary and conclusion

Conclusions:

ABSORPTION
- For Group 1 male rats orally dosed with [14C] test item, the peak of radioactivity or Tmax in plasma was observed at 2 hours postdose. The mean peak concentration in plasma or Cmax at 2 hours was 1,644.89 ± 460.50 ng-eq/g. The Cmax was followed by a slight decline through 8 hours postdose (Distribution Phase), and a steeper decline thereafter (Elimination Phase). The last measurable mean plasma value at 48 hours postdose was 22.91 ± 45.81 ng-eq/g. The plasma value at 72 hours postdose did not have measurable levels of test article, and was recorded as zero.
- For Group 2 female rats orally dosed with [14C] test item, the peak of radioactivity or Tmax in plasma was observed at 1 hour postdose. The mean peak concentration in plasma or Cmax at 1 hour was 1,850.39 ± 431.95 ng-eq/g. The Cmax was followed by a steady decline through 72 hours postdose, and distinct Distribution and Elimination phases were indistinguishable. The last measurable mean plasma value at 72 hours postdose was 38.67 ± 77.34 ng-eq/g.
- For Group 5 male rats orally dosed with [14C] test item, terminal blood was removed and analyzed following euthanasia at all QWBA timepoints. The plasma values for this group were single point analyses, and statistical analysis was not possible. Plasma concentration values in this group correlated well with the plasma concentration values obtained for Group 1 orally dosed male rats.
- For Group 6 female rats orally dosed with [14C] test item, terminal blood was removed and analyzed following euthanasia at all QWBA timepoints. The plasma values for this group were single point analyses, and statistical analysis was not possible. Plasma concentration values in this group correlated well with the plasma concentration values obtained for Group 2 orally dosed female rats.
- Blood to plasma ratios were calculated for all animals in all groups where blood was sampled. For Group 1 males, blood to plasma ratios ranged from a mean average of 0.62 to 0.86 through 24 hours postdose. These values are considered normal, indicative of low partitioning to the erythrocytes. At 48 hours postdose, the ratio could only be calculated for one animal (104M), which was 1.95 – indicative of higher partitioning to the erythrocytes. At 72 hours postdose, the blood to plasma ration could not be calculated, as the plasma values were recorded as zero. For Group 2 females, mean blood to plasma ratios ranged from 0.68 to 0.87 through 8 hours postdose, after which mean values rose to 1.57 at 24 hours postdose, and 5.48 at 48 hours postdose, indicative of high partitioning to erythrocytes with increased time. Blood to plasma ratios for both male and females for both Groups 5 and 6 (QWBA animals) followed the same pattern outlined for Groups 1 and 2 males and females.

DISTRIBUTION
- Following oral administration of [14C] test item to intact male and female rats, the test substance-derived radioactivity was widely distributed in both sexes. In general, distribution was faster in females, as evidenced by a Cmax of 2 hours for females versus 8 hours for males. Additionally, the quantitative levels in certain common tissues were higher for females than males.
- The limit of quantitation was determined to be approximately 382 nanogram-equivalents of [14C] test item per gram of tissue. A concentration of 2,000 ng-eq/g was logically selected as a threshold value representing significant exposure to the tissues analysed (based on the results obtained). At this selected threshold value, 16 of 34 tissues quantitated were noted as above threshold at the observed tissue Cmax of 8 hours postdose in males, and 18 of 34 tissues quantitated were noted as above threshold at the observed tissue Cmax of 2 hours postdose in females. As would be expected for an orally administered test article, the majority of the tissues noted as positive are part of the gastrointestinal tract (and associated organ systems), for both males and females.
- For intact male rats, the 16 tissues noted with concentrations greater than the selected threshold value of 2,000 ng-eq/g at 8 hours included (listed from highest to lowest concentration): Stomach contents (2,071,270 ng-eq/g); Cecum contents (1,020,869 ng-eq/g); Large intestinal contents (33,968 ng-eq/g); Liver (14,449 ng-eq/g); Adrenal gland (9,686 ng-eq/g); Kidney (5,026 ng-eq/g); Fat, brown (3,854 ng-eq/g); Pancreas (3,838 ng-eq/g); Thyroid gland (3,229 ng-eq/g); Cecum (2,982 ng-eq/g); Salivary gland (2,799 ng-eq/g); Lung (2,680 ng-eq/g); Myocardium (2,647 ng-eq/g); Skin (2,560 ng-eq/g); Spleen (2,431 ng-eq/g); and Large intestine (2,008 ng-eq/g). Several tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 2 hours postdose (the first QWBA timepoint) included (listed from highest to lowest concentration): Small intestinal contents (882,343 ng-eq/g); Small intestine (18,998 ng-eq/g); Urine (12,447 ng-eq/g); Urinary bladder (4,993 ng-eq/g); and Stomach (2,733 ng-eq/g).
- Other tissues in the males where distribution was noted as positive, but that was less than the selected threshold included: Bone (647 ng-eq/g only observed at 168 hours postdose); Cerebellum (705 ng-eq/g only observed at 2 hours postdose); Medulla (670 ng-eq/g only observed at 2 hours postdose); Bone marrow (Cmax of 1,660 ng-eq/g at 8 hours postdose); Prostate gland (Cmax of 1,600 ng-eq/g at 8 hours postdose); Thymus (Cmax of 1,551 ng-eq/g at 8 hours postdose); Muscle (Cmax of 1,251 ng-eq/g at 8 hours postdose); Blood (Cmax of 1,087 ng-eq/g at 8 hours postdose); Testes (Cmax of 1,084 ng-eq/g at 8 hours postdose); Fat, white (Cmax of 688 ng-eq/g at 2 hours postdose); Olfactory lobe (Cmax of 501 ng-eq/g at 2 hours postdose); Cerebrum (Cmax of 681 ng-eq/g at 2 hours postdose); and Eye, lens (382 ng-eq/g only observed at 8 hours postdose).
- For intact female rats, the 18 tissues noted with concentrations greater than the threshold value of 2,000 ng-eq/g at 2 hours included (listed from highest to lowest concentration): Stomach contents (1,261,640 ng-eq/g); Small intestinal contents (1,077,609 ng-eq/g); Small intestine (29,410 ng-eq/g); Liver (17,158 ng-eq/g); Stomach (17,068 ng-eq/g); Adrenal gland (14,986 ng-eq/g); Kidney (6,590 ng-eq/g); Salivary gland (6,329 ng-eq/g); Thyroid gland (5,492 ng-eq/g); Fat, brown (5,384 ng-eq/g); Pancreas (5,110 ng-eq/g); Lung (4,281 ng-eq/g); Spleen (3,194 ng-eq/g); Myocardium (3,143 ng-eq/g); Uterus (2,746 ng-eq/g); Olfactory lobe (2,135 ng-eq/g); Thymus (2,135 ng-eq/g); and Bone marrow (2,068 ng-eq/g). Several tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 8 hours postdose (the second QWBA timepoint) included (listed from highest to lowest concentration): Large intestinal contents (1,447,956 ng-eq/g); Cecum contents (335,591 ng-eq/g); Cecum (12,183 ng-eq/g); Eye, lens (2,855 ng-eq/g); and Large intestine (2,736 ng-eq/g). Two tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 24 hours postdose (the third QWBA timepoint) included (listed from highest to lowest concentration): Skin (4,577 ng-eq/g); and Ovary (3,332 ng-eq/g).
- Other tissues in the females where distribution was noted that was less than threshold included: Bone (1,579 ng-eq/g only observed at 168 hours postdose); Cerebellum (1,489 ng-eq/g Cmax observed at 2 hours postdose); Medulla (1,399 ng-eq/g Cmax observed at 2 hours postdose); Muscle (Cmax of 1,408 ng-eq/g at 2 hours postdose); Blood (Cmax of 1,315 ng-eq/g at 2 hours postdose); Fat, white (Cmax of 1,625 ng-eq/g at 2 hours postdose); and Cerebrum (Cmax of 1,530 ng-eq/g at 2 hours postdose).

METABOLISM
- The [14C] test item starting material is a mixture of components which is extensively metabolized, with little to none of the starting material observed in any of the matrices examined in this study.
- The major metabolite noted in feces of males, greater than five percent of dose, was M39, which was 23.8% of dose at 8-24 hours, and 11.6% of dose at 24-48 hours postdose. In females, the M39 was 14.5% of dose at 24-48 hours, 12.9% of dose at 24-48 hours, and 5.4% of dose at 48-72 hours postdose.
- The major metabolite noted in bile of males, greater than five percent of dose, was also M39, which was 4.4% of dose at 0-8 hours, 8.4% at 8-24 hours, and 2.0% of dose at 24- 48 hours postdose. Bile collection from bile-duct canulated female rats was not part of the study design.
- Metabolite M39 was not observed in urine from males, but was observed in urine from females at 0.2% of dose, only in the 8-24 hour postdose sample. It should also be noted that all the metabolites characterized were less than one percent of dose in urine, which is not the major route of excretion for this test material.
- Metabolite M39 was also observed in plasma from 2, 8, and 24 hours postdose pooled plasma at 62.1% ROI in males and 68.6% ROI in females. It should be noted that with plasma, only a percent region of interest (% ROI) can be calculated, and not a percent of dose.

ELIMINATION
- For Group 1 orally dosed male rats, the main route of elimination of [14C] test item was fecal excretion. The mean fecal recovery of the radioactive dose through 168 hours postdose was 71.93% ± 2.82%. Zero percent of the total dose recovered from the feces was recovered within the first 8 hours postdose, and approximately 75.03% of the total dose recovered from the feces was recovered from 8-24 hours postdose. A total of 97.37% of the total dose recovered from the feces was recovered within the first 48 hours postdose.
- Urinary excretion of [14C] test item was relatively low in male animals, with a total recovery of just 4.25% ± 0.70% of the total dose recovered through 168 hours postdose.
- Mean total recovery of [14C] test item from carcasses at 168 hours postdose was 0.87% ± 0.19% for orally dosed male rats.
- For Group 1 male rats, mean total recovery of the orally administered radioactive dose of [14C] test item from all sources through 168 hours postdose was 78.02% ± 2.56%, with individual total recovery values of: 76.88%, 77.86%, 75.71%, and 81.62%.
- The radioactive material unaccounted for represents approximately 21.98% of the total orally administered dose of [14C] test item to Group 1 male rats.
- For Group 2 orally dosed female rats, the main route of elimination of [14C] test item was also fecal excretion. The mean fecal recovery of the radioactive dose through 168 hours postdose was 76.00% ± 10.43%. Just 0.30% of the total dose recovered from the feces was recovered within the first 8 hours postdose, and approximately 55.46% of the total dose recovered from the feces was recovered from 8-24 hours postdose. A total of 85.49% of the total dose recovered from the feces was recovered within the first 48 hours postdose. The mean percentage of the radioactive dose collected in the feces of females was slightly greater than males (76.00% versus71.93%), and the rate of fecal elimination was slower than the rate observed in males.
- Urinary excretion of [14C] test item was relatively low in female animals, with a total recovery of just 4.85% ± 0.20% of the total dose recovered through 168 hours postdose. The mean percentage recovery in urine for females compares well with that for males (4.85% versus 4.25%).
- Mean total recovery of [14C]test item from carcasses at 168 hours postdose was 1.62% ± 0.62% – nearly double that observed for males (1.62% versus 0.87%).
- For Group 2 female rats, mean total recovery of the orally administered radioactive dose of [14C] test item from all sources through 168 hours postdose was 83.70% ± 9.75%, with individual total recovery values of: 82.64%, 72.94%, 96.63%, and 82.60%. The mean total recovery was better for females (83.70% versus 71.93%).
- The radioactive material unaccounted for represents approximately 16.30% of the total orally administered dose of [14C] test item, which was slightly better than the value observed for males (21.98%).
- Biliary excretion of [14C] test item in Group 3 male rats was significant, with a cumulative mean recovery of 31.06% ± 2.44% of the total dose administered recovered through 72 hours postdose. Recovery of the bile in individual animals was consistent, ranging from a low of 27.55% to a high of 32.91%. The majority of the radioactivity in bile-duct canulated male animals was observed in the bile from 0-8 hours, with approximately 51.19% of the total dose recovered from the bile obtained in the first 8 hours, and 92.56% of the total dose collected from the bile recovered within the first 24 hours postdose.
- Excretion of [14C] test item in expired air in Group 4 male rats over 72 hours postdose was zero percent via this excretion route.

Executive summary:

INTRODUCTION

The study was conducted to determine the plasma time-course, routes and rates of excretion with mass balance, tissue distribution, and metabolite profiling of radioactivity in male and female Sprague-Dawley rats, following administration of a single oral dose of [14C] test item. The method complied with the OECD 417 guidelines, EPA OPPTS 870.7485, requirements contained in the MPI Research Radioactive Materials License Number 21-11315-02, and all applicable regulations issued by the Nuclear Regulatory Commission (NRC).

 

METHODS

Animals were assigned to the study as described in the following table.

 

Group number	Average dose concentration (mg/kg)	Average dose radioactivity (µCi/kg)	Target dose volume (mL/kg)	Samples collected	Number of animals
1	28.653	177.0	5	Blood, plasma, urine, feces, cage rinses, cage wipes and carcasses	4 males (JVC)
2	29.386	181.5	5	Blood, plasma, urine, feces, cage rinses, cage wipes and carcasses	4 females (JVC)
3	29.615	182.9	5	Bile	4 Males (BDC)
4	29.330	181.2	5	Expired air	4 males
5	29.873	184.5	5	Terminal blood, plasma and carcasses for QWBA	5 males
6	30.034	185.5	5	Terminal blood, plasma and carcasses for QWBA	5 females

BDC = Bile duct cannulated

JVC = Jugular vein cannulated

QWBA = Quantitative whole body radiography

 

Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Body weights were measured and recorded prior to dosing (Day -1). Blood samples were collected at designated time points up to 72 (Groups 1 and 2) or 168 (Groups 5 and 6) hours postdose for whole blood and plasma radioactivity analysis. Urine, feces, and cage residue were collected from Groups 1 and 2 at designated intervals up to 168 hours postdose for radioactivity concentration analysis. Bile and expired air were collected from Group 3 and 4 animals, respectively up to 72 hours postdose for radioactivity concentration analysis. At the terminal time points for each animal in Groups 1 and 2, the animals were euthanized and carcasses were collected and analyzed for concentration of radioactivity. At designated time points postdose following euthanasia, the Group 5 and 6 animal carcasses were prepared and examined via quantitative whole body autoradiography (QWBA) analysis. At study termination, all animals in Groups 3 and 4 were euthanized and the carcasses were stored frozen pending disposal. Following radioanalysis, metabolite profiling was performed on plasma, urine, feces, and bile samples.

 

ADSORPTION

For Group 1 male rats orally dosed with [14C] test item, the peak of radioactivity or Tmax in plasma was observed at 2 hours postdose. The mean peak concentration in plasma or Cmax at 2 hours was 1,644.89 ± 460.50 ng-eq/g. The Cmax was followed by a slight decline through 8 hours postdose (Distribution Phase), and a steeper decline thereafter (Elimination Phase). The last measurable mean plasma value at 48 hours postdose was 22.91 ± 45.81 ng-eq/g. The plasma value at 72 hours postdose did not have measurable levels of test article, and was recorded as zero.

 

For Group 2 female rats orally dosed with [14C] test item, the peak of radioactivity or Tmax in plasma was observed at 1 hour postdose. The mean peak concentration in plasma or Cmax at 1 hour was 1,850.39 ± 431.95 ng-eq/g. The Cmax was followed by a steady decline through 72 hours postdose, and distinct Distribution and Elimination phases were indistinguishable. The last measurable mean plasma value at 72 hours postdose was 38.67 ± 77.34 ng-eq/g.

 

For Group 5 male rats orally dosed with [14C] test item, terminal blood was removed and analyzed following euthanasia at all QWBA timepoints. The plasma values for this group were single point analyses, and statistical analysis was not possible. Plasma concentration values in this group correlated well with the plasma concentration values obtained for Group 1 orally dosed male rats.

 

For Group 6 female rats orally dosed with [14C] test item, terminal blood was removed and analyzed following euthanasia at all QWBA timepoints. The plasma values for this group were single point analyses, and statistical analysis was not possible. Plasma concentration values in this group correlated well with the plasma concentration values obtained for Group 2 orally dosed female rats.

 

Blood to plasma ratios were calculated for all animals in all groups where blood was sampled. For Group 1 males, blood to plasma ratios ranged from a mean average of 0.62 to 0.86 through 24 hours postdose. These values are considered normal, indicative of low partitioning to the erythrocytes. At 48 hours postdose, the ratio could only be calculated for one animal (104M), which was 1.95 – indicative of higher partitioning to the erythrocytes. At 72 hours postdose, the blood to plasma ration could not be calculated, as the plasma values were recorded as zero. For Group 2 females, mean blood to plasma ratios ranged from 0.68 to 0.87 through 8 hours postdose, after which mean values rose to 1.57 at 24 hours postdose, and 5.48 at 48 hours postdose, indicative of high partitioning to erythrocytes with increased time. Blood to plasma ratios for both male and females for both Groups 5 and 6 (QWBA animals) followed the same pattern outlined for Groups 1 and 2 males and females.

 

DISTRIBUTION

Following oral administration of [14C] test item to intact male and female rats, the test substance-derived radioactivity was widely distributed in both sexes. In general, distribution was faster in females, as evidenced by a Cmax of 2 hours for females versus 8 hours for males. Additionally, the quantitative levels in certain common tissues were higher for females than males.

 

The limit of quantitation was determined to be approximately 382 nanogram-equivalents of [14C] test item per gram of tissue. A concentration of 2,000 ng-eq/g was logically selected as a threshold value representing significant exposure to the tissues analysed (based on the results obtained). At this selected threshold value, 16 of 34 tissues

quantitated were noted as above threshold at the observed tissue Cmax of 8 hours postdose in males, and 18 of 34 tissues quantitated were noted as above threshold at the observed tissue Cmax of 2 hours postdose in females. As would be expected for an orally administered test article, the majority of the tissues noted as positive are part of the gastrointestinal tract (and associated organ systems), for both males and females.

 

For intact male rats, the 16 tissues noted with concentrations greater than the selected threshold value of 2,000 ng-eq/g at 8 hours included (listed from highest to lowest concentration): Stomach contents (2,071,270 ng-eq/g); Cecum contents (1,020,869 ng-eq/g); Large intestinal contents (33,968 ng-eq/g); Liver (14,449 ng-eq/g); Adrenal gland (9,686 ng-eq/g); Kidney (5,026 ng-eq/g); Fat, brown (3,854 ng-eq/g); Pancreas (3,838 ng-eq/g); Thyroid gland (3,229 ng-eq/g); Cecum (2,982 ng-eq/g); Salivary gland (2,799 ng-eq/g); Lung (2,680 ng-eq/g); Myocardium (2,647 ng-eq/g); Skin (2,560 ng-eq/g); Spleen (2,431 ng-eq/g); and Large intestine (2,008 ng-eq/g). Several tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 2 hours postdose (the first QWBA timepoint) included (listed from highest to lowest concentration): Small intestinal contents (882,343 ng-eq/g); Small intestine (18,998 ng-eq/g); Urine (12,447 ng-eq/g); Urinary bladder (4,993 ng-eq/g); and Stomach (2,733 ng-eq/g).

 

Other tissues in the males where distribution was noted as positive, but that was less than the selected threshold included: Bone (647 ng-eq/g only observed at 168 hours postdose); Cerebellum (705 ng-eq/g only observed at 2 hours postdose); Medulla (670 ng-eq/g only observed at 2 hours postdose); Bone marrow (Cmax of 1,660 ng-eq/g at 8 hours postdose); Prostate gland (Cmax of 1,600 ng-eq/g at 8 hours postdose); Thymus (Cmax of 1,551 ng-eq/g at 8 hours postdose); Muscle (Cmax of 1,251 ng-eq/g at 8 hours postdose); Blood (Cmax of 1,087 ng-eq/g at 8 hours postdose); Testes (Cmax of 1,084 ng-eq/g at 8 hours postdose); Fat, white (Cmax of 688 ng-eq/g at 2 hours postdose); Olfactory lobe (Cmax of 501 ng-eq/g at 2 hours postdose); Cerebrum (Cmax of 681 ng-eq/g at 2 hours postdose); and Eye, lens (382 ng-eq/g only observed at 8 hours postdose).

 

For intact female rats, the 18 tissues noted with concentrations greater than the threshold value of 2,000 ng-eq/g at 2 hours included (listed from highest to lowest concentration): Stomach contents (1,261,640 ng-eq/g); Small intestinal contents (1,077,609 ng-eq/g); Small intestine (29,410 ng-eq/g); Liver (17,158 ng-eq/g); Stomach (17,068 ng-eq/g); Adrenal gland (14,986 ng-eq/g); Kidney (6,590 ng-eq/g); Salivary gland (6,329 ng-eq/g); Thyroid gland (5,492 ng-eq/g); Fat, brown (5,384 ng-eq/g); Pancreas (5,110 ng-eq/g); Lung (4,281 ng-eq/g); Spleen (3,194 ng-eq/g); Myocardium (3,143 ng-eq/g); Uterus (2,746 ng-eq/g); Olfactory lobe (2,135 ng-eq/g); Thymus (2,135 ng-eq/g); and Bone marrow (2,068 ng-eq/g). Several tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 8 hours postdose (the second QWBA timepoint) included (listed from highest to lowest concentration): Large intestinal contents (1,447,956 ng-eq/g); Cecum contents (335,591 ng-eq/g); Cecum (12,183 ng-eq/g); Eye, lens (2,855 ng-eq/g); and Large intestine (2,736 ng-eq/g). Two tissues that had concentrations greater than the selected threshold, but had their respective Cmax at 24 hours postdose (the third QWBA timepoint) included (listed from highest to lowest concentration): Skin (4,577 ng-eq/g); and Ovary (3,332 ng-eq/g).

 

Other tissues in the females where distribution was noted that was less than threshold included: Bone (1,579 ng-eq/g only observed at 168 hours postdose); Cerebellum (1,489 ng-eq/g Cmax observed at 2 hours postdose); Medulla (1,399 ng-eq/g Cmax observed at 2 hours postdose); Muscle (Cmax of 1,408 ng-eq/g at 2 hours postdose); Blood (Cmax of 1,315 ng-eq/g at 2 hours postdose); Fat, white (Cmax of 1,625 ng-eq/g at 2 hours postdose); and Cerebrum (Cmax of 1,530 ng-eq/g at 2 hours postdose).

 

METABOLISM

The [14C] test item starting material is a mixture of components which is extensively metabolized, with little to none of the starting material observed in any of the matrices examined in this study.

 

The major metabolite noted in feces of males, greater than five percent of dose, was M39, which was 23.8% of dose at 8-24 hours, and 11.6% of dose at 24-48 hours postdose. In females, the M39 was 14.5% of dose at 24-48 hours, 12.9% of dose at 24-48 hours, and 5.4% of dose at 48-72 hours postdose.

 

The major metabolite noted in bile of males, greater than five percent of dose, was also M39, which was 4.4% of dose at 0-8 hours, 8.4% at 8-24 hours, and 2.0% of dose at 24-48 hours postdose. Bile collection from bile-duct canulated female rats was not part of the study design.

 

Metabolite M39 was not observed in urine from males, but was observed in urine from females at 0.2% of dose, only in the 8-24 hour postdose sample. It should also be noted that all the metabolites characterized were less than one percent of dose in urine, which is not the major route of excretion for this test material.

 

Metabolite M39 was also observed in plasma from 2, 8, and 24 hours postdose pooled plasma at 62.1% ROI in males and 68.6% ROI in females. It should be noted that with plasma, only a percent region of interest (% ROI) can be calculated, and not a percent of dose.

 

ELIMINATION

For Group 1 orally dosed male rats, the main route of elimination of [14C] test item was fecal excretion. The mean fecal recovery of the radioactive dose through 168 hours postdose was 71.93% ± 2.82%. Zero percent of the total dose recovered from the feces was recovered within the first 8 hours postdose, and approximately 75.03% of the total dose recovered from the feces was recovered from 8-24 hours postdose. A total of 97.37% of the total dose recovered from the feces was recovered within the first 48 hours postdose.

 

Urinary excretion of [14C] test item was relatively low in male animals, with a total recovery of just 4.25% ± 0.70% of the total dose recovered through 168 hours postdose.

 

Mean total recovery of [14C] test item from carcasses at 168 hours postdose was 0.87% ± 0.19% for orally dosed male rats.

 

For Group 1 male rats, mean total recovery of the orally administered radioactive dose of [14C] test item from all sources through 168 hours postdose was 78.02% ± 2.56%, with individual total recovery values of: 76.88%, 77.86%, 75.71%, and 81.62%.

 

The radioactive material unaccounted for represents approximately 21.98% of the total orally administered dose of [14C] test item to Group 1 male rats.

 

For Group 2 orally dosed female rats, the main route of elimination of [14C] test item was also fecal excretion. The mean fecal recovery of the radioactive dose through 168 hours postdose was 76.00% ± 10.43%. Just 0.30% of the total dose recovered from the feces was recovered within the first 8 hours postdose, and approximately 55.46% of

the total dose recovered from the feces was recovered from 8-24 hours postdose. A total of 85.49% of the total dose recovered from the feces was recovered within the first 48 hours postdose. The mean percentage of the radioactive dose collected in the feces of females was slightly greater than males (76.00% versus71.93%), and the rate of fecal elimination was slower than the rate observed in males.

 

Urinary excretion of [14C] test item was relatively low in female animals, with a total recovery of just 4.85% ± 0.20% of the total dose recovered through 168 hours postdose. The mean percentage recovery in urine for females compares well with that for males (4.85% versus 4.25%).

 

Mean total recovery of [14C] test item from carcasses at 168 hours postdose was 1.62% ± 0.62% – nearly double that observed for males (1.62% versus 0.87%).

 

For Group 2 female rats, mean total recovery of the orally administered radioactive dose of [14C] test item from all sources through 168 hours postdose was 83.70% ± 9.75%, with individual total recovery values of: 82.64%, 72.94%, 96.63%, and 82.60%. The mean total recovery was better for females (83.70% versus 71.93%).

 

The radioactive material unaccounted for represents approximately 16.30% of the total orally administered dose of [14C] test item, which was slightly better than the value observed for males (21.98%).

 

Biliary excretion of [14C] test item in Group 3 male rats was significant, with a cumulative mean recovery of 31.06% ± 2.44% of the total dose administered recovered through 72 hours postdose. Recovery of the bile in individual animals was consistent, ranging from a low of 27.55% to a high of 32.91%. The majority of the radioactivity in bile-duct canulated male animals was observed in the bile from 0-8 hours, with approximately 51.19% of the total dose recovered from the bile obtained in the first 8 hours, and 92.56% of the total dose collected from the bile recovered within the first 24 hours postdose.

 

Excretion of [14C] test item in expired air in Group 4 male rats over 72 hours postdose was zero percent via this excretion route.