1,2,4-Oxadiazole, 3-phenyl-5-(2-thienyl)-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 27, 2011 to July 2, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 417 and OPPTS 870.7485, in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-phenyl-5-(thiophen-2-yl)-1,2,4-oxadiazole

EC Number:

810-533-8

Cas Number:

330459-31-9

Molecular formula:

C12H8N2OS

IUPAC Name:

3-phenyl-5-(thiophen-2-yl)-1,2,4-oxadiazole

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): MON 102100
- Two independently labelled forms of [14C]-MON 102100 (designated PH and TH) were utilized in this study

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: Seven to ten weeks old
- Weight at study initiation: 207-289 g (male) and 186-248 g (female)
- Housing: Housed individually in clean, suspended wire-mesh cages
- Individual metabolism cages: Yes
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was offered ad libitum during the acclimation and the biological phases of the study.
- Water: Reverse osmosis treated water was provided ad libitum
- Acclimation period: 1 week (non-cannulated animals) and 2 d (bile duct-cannulated (BDC) animals)

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C
- Humidity: 50% ± 20%
- Air changes: 10 air changes/h
- Photoperiod: 12 h of fluorescent light followed by 12 h of darkness cycle. The light/dark cycle was interrupted for protocol-specified activities

Administration / exposure

Route of administration:

other: Oral gavage and intravenous

Vehicle:

other: 0.5% CMC (Oral gavage); EtOH:Cremophor (1:1) and 0.9% sodium chloride for injection USP (intravenous)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Storage: Formulations were stored refrigerated (approximately 2°C to 8°C)

Duration and frequency of treatment / exposure:

Single dose of radiolabeled substance.
Animals in three groups received daily doses of non-labelled test substance at 3 mg/kg bw/day for 14 d prior to the radiolabeled dose administration.
Refer to overall study design is summarized in Table 1 under ‘Any other information on materials and methods incl. tables’ section.

Doses / concentrations

Remarks:

Doses / Concentrations:
This study was conducted in multiple phases: a Pilot Phase (Groups 1A and 1B), a Pharmacokinetic Phase (Groups 2A, 2B, 3A, 3B, 4A, and 4B), a Disposition and Metabolite Identification Phase (Groups 5A, 5B, 6A, 6B, 7A, 7B, 8A, 8B, 11A, 11B, 12A, and 13A), and a Quantitative Whole Body Autoradiography (QWBA) Phase (Groups 9A, 9B, 10A, and 10B).

The overall study design is summarized in Table 1 under ‘Any other information on materials and methods incl. tables’ section.

No. of animals per sex per dose / concentration:

- Refer to Table 1 under ‘Any other information on materials and methods incl. tables’ section

- Pilot phase groups: 2 males/group

- Pharmacokinetic phase: 8 males/group

- Disposition and metabolite identification phase: 4 males/group. Additional 4 females were included in group 6A, 6B and 12 A

- QWBA phase: 4 animals/sex/group

Control animals:

no

Details on study design:

- Dose selection rationale: Dose levels were selected based on the previous 28 d and 90 d rat oral toxicology studies with test substance. The low dose level of 3 mg/kg bw was expected to be non toxic; the high dose level of 100 mg/kg is a dose level at which some toxicity and/or changes in toxicokinetic parameters would be expected following repeat exposures.

On the day prior to dosing, non-cannulated animals judged to be suitable for testing were weighed and assigned to the groups at random using a computer program. BDC animals were assigned to study on the day of dosing based on the overnight bile production animals with the greatest volume of bile were selected.

Details on dosing and sampling:

The Pilot Phase groups consisted of two male Crl:CD(SD) rats per test substance. Each animal received a single oral (gavage) dose of [PH-U-14C]-test substance or [TH-2-14C]-test substance at a target dose of 3 mg/kg bw. Following dosing, all animals were placed into individual glass metabolism units (Roth type), for separate collection of expired air components, urine, and faeces through 48 h post-dosing. After the final excreta collection at approximately 48 h post-dosing, animals were euthanized by CO2 inhalation and carcasses discarded.

The Pharmacokinetic Phase consisted of six groups of eight male rats each. For both [PH-U-14C]-test substance and [TH-2-14C]-test substance, each animal received either a single IV (bolus) dose at 3 mg/kg bw, a single oral (gavage) dose at 3 mg/kg bw, or a single oral (gavage) dose at 100 mg/kg bw. Following dosing, all animals were returned to their wire mesh cages. Blood samples were collected from four animals/group/time point alternating between two sub groups in each group. Blood samples were collected following IV dosing at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, and 96 h post dosing. Blood samples were collected following oral dosing at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, and 168 h post dosing. Immediately following the final blood collection, animals were euthanized by CO2 inhalation and the carcasses were discarded. Blood was isolated to plasma in a refrigerated centrifuge and plasma was analysed for total radioactivity.

The Disposition and Metabolite Identification Phase consisted of 12 groups of four male and three groups of four female Crl:CD(SD) rats. For both [PH-U-14C]-labeled test substance and [TH-2-14C]-labeled test substance, male rats received a single IV (bolus) dose at 3 mg/kg bw, a single oral (gavage) dose at 3 mg/kg bw, or a single oral (gavage) dose at 100 mg/kg bw Female rats received a single oral (gavage) dose at 3 mg/kg bw. Additional male rats received a daily oral (gavage) dose of non-labelled test substance at 3 mg/kg bw for 14 consecutive days followed by a single oral (gavage) dose of [PH-U-14C]-labeled test substance or [TH-2-14C]-labeled test substance at 3 mg/kg bw. Furthermore, male bile duct-cannulated (BDC) rats received a single oral (gavage) dose of [PH-U-14C]-lebeled test substance or [TH-2-14C]-labeled test substance at 100 mg/kg bw. Initially, quantitation of the administered radiolabeled dose was determined by weighing each dosing syringe before and after delivery of the dose. However, due to apparently low recovery, the experiments with the three groups receiving either a single oral dose (males and females) or a repeated non-radiolabeled dose followed by a single oral dose (males only) of [PH-U-14C]-labeled test substace at 3 mg/kg bw were conducted a second time; for these groups, the actual amount of the delivered dose was further determined by rinsing the dosing syringe with MeOH and determining the total radioactivity that was retained in the syringe after the dispensing of the dose. Following dosing, all animals in the Disposition and Metabolite Identification Phases were placed in individual plastic metabolism cages for separate collection of urine and faeces through 168 h post-dosing. At approximately 168 h post-dosing, after the final excreta collection, animals were euthanized by CO2 inhalation and weighed. Blood, select tissues, and the remaining carcass were collected and analysed for total radioactivity. Bile, urine, and faeces were collected over 48 h post-dosing from BDC animals. Based on total radioactivity results, selected urine and bile samples and faecal extracts were pooled and profiled for metabolites using radio-HPLC.

The QWBA Phase consisted of four groups of four male and four female Crl:CD(SD) each. Animals received a single oral (gavage) dose of [PH-U-14C]- labeled test substance or [TH-2-14C]- labeled test substance at target doses of 3 mg/kg bw or 100 mg/kg bw. Following dosing, animals were returned to their individual wire mesh cages. At 2 h (plasma Tmax) and 48 h post-dosing for animals dosed at 3 mg/kg bw, two animals/sex were anesthetized with isoflurane and a blood sample was collected via cardiac puncture. At 4 h (plasma Tmax) and 48 h post-dosing for animals dosed at 100 mg/kg bw, two animals/sex were anesthetized with isoflurane and a blood sample was collected via cardiac puncture. Following blood collection, each anesthetized animal was euthanized by CO2 inhalation, submerged in a dry ice/hexane bath, and the frozen carcasses were embedded in CMC for QWBA analysis of 26 tissues in females and 27 tissues in males.

Statistics:

Calculations of the amounts of radioactivity in various materials generated in the study will be performed using Microsoft Excel 2007 software. Generally, only descriptive statistics (e.g., totals, arithmetic means, standard deviations, standard errors, coefficients of variation, percentages) will be used. Pharmacokinetic parameters will be calculated from the mean plasma concentration values for the total radioactivity. The pharmacokinetic parameters that will be calculated include, but are not limited to: for the IV dosing: area under the plasma concentration vs. time curve (AUC), half-life; clearance and apparent volume of distribution; for the oral dosing: C, t, AUC, half-life and bioavailability. Calculations will be performed using validated WinNonLin 5.2 software (Pharsight Corp., Mountain View, CA). Graphs will be prepared, when necessary, to illustrate the salient features of the pharmacokinetic data.

Results and discussion

Preliminary studies:

Pooled urine samples and extracts of pooled faecal homogenates indicated significant differences in the metabolite profiles for rats receiving either [PH-U-14C] test substance or [TH-2-14C] test substance. Thus, both radiolabels were dosed for all definitive groups in the study. Radioactivity in expired air was <1.0% of the administered dose for both radiolabels; therefore, collection of expired air components was not conducted for the disposition and metabolite identification phase

Main ADME results

Type:

other:

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Bioavailability (percent oral absorption) calculated from the IV and the oral administration at the same dose was approximately 58% for the [PH-U-14C] test substance label and 73% for the [TH-2-14C] test substance. At the 100 mg/kg bw, the mean terminal phase halflife (T1/2) of [14C] test substance equivalents in plasma was approximately 44 to 47 h following the low oral dose.

At the high oral dose (100 mg/kg bw), the plasma Cmax values were 23 to 25 μg/g (Tmax = 4 h); the overall exposure (AUCinf) was 543 to 587 μg•h/g and may have been slightly more than dose proportionate; however, data obtained during the disposition and metabolite identification phase (groups 6A and 8A) indicated that delivery of the low doses for the PH label may have been below their target due to test substance binding to the dosing syringe, affecting the evaluation of dose proportionality. The mean terminal phase half life (T1/2) of [14C] test substance equivalents in plasma was approximately 38 to 42 h following the high oral dose.

Details on distribution in tissues:

The QWBA data generally reflected the trend that test substance was widely distributed and that the concentration of [14C] test substance equivalents rapidly declined over the 48 h collection period. Across dose levels (3 mg/kg or 100 mg/kg) and collection times (Tmax or 48 h), tissue-to-plasma ratios were generally significantly greater than unity in adrenal gland, liver, kidney, kidney (cortex), thyroid gland, stomach and small intestine (at Tmax), fat (100 mg/kg dose), and urinary bladder. At Tmax (2 h post-dosing), the highest concentrations following a 3 mg/kg dose were in the stomach, kidney (cortex), liver, total kidney (mean concentration across the entire organ), and adrenal gland (generally >2.5 μg/g), and then the bladder, kidney (medulla), harderian gland, small intestine, thyroid, and lung (generally >0.5 μg/g). There appeared to be a sex difference in the stomach concentration, 9 μg/g to 26 μg/g in males versus 0.8 μg/g to 5.6 μg/g in females at Tmax. The lowest concentrations were in the eye and bone (<0.1 μg/g).

Forty-eight h after dosing at 3 mg/kg, the concentration had generally decreased to ≤1.6 μg/g for the adrenal gland, <0.5 μg/g for the next highest tissues (kidney and kidney cortex, liver, urinary bladder, and thyroid), and <0.1 μg/g for the remaining tissues (both labels). At Tmax (4 h post-dosing), the highest concentrations following a 100 mg/kg dose were in the adrenal gland and urinary bladder of males and females after [PH-U-14C]-test substance administration, and in the adrenal gland, kidney (cortex), liver, and stomach of males and adrenal gland and fat of females after [TH-2-14C]-test substance administration (generally 100 μg/g to 500 μg/g). The concentrations in harderian gland, small intestine, thyroid, kidney, fat, and liver in other groups were generally between 30 μg/g to 80 μg/g. For the remaining tissues, the concentration was generally <30 μg/g. The lowest concentrations were in the eye and bone (<5 μg/g). Forty-eight h after dosing at 100 mg/kg, the concentration had generally decreased to ≤70 μg/g for the adrenal gland, <15 μg/g (PH label) or 20 μg/g (TH label) for the next highest tissues (liver, kidney, kidney [cortex], thyroid), and <7.5 μg/g for the remaining tissues.

Details on excretion:

In all Disposition and Metabolite Identification groups (excluding groups repeated due to low recovery and BDC animals), [14C]-test substance equivalents were excreted primarily in the faeces, where an average of 45% to 69% of the administered dose was recovered, and in urine, where an average of 24% to 38% of the dose was recovered. Of the dose recovered (excreted) in the urine and faeces, about one-third (mean of 35%, range of 30% to 43%) was excreted in urine and about two-thirds (mean of 65%, range of 57% to 70%) was excreted in faeces. In general, there was no trend in excretion pattern related to the position of the radiolabel, the dose level or the dose route, single or repeated administration, or the sex of the rats. There was possibly a slight difference in the pattern of excretion comparing the IV PH- and TH-label doses for which urinary elimination was 43% and 33% of the eliminated dose, respectively. The average total recovery of [14C]-test substance equivalents across all groups (excluding groups repeated due to low recovery) was 93% of the administered dose. The greatest fraction of the administered dose recovered in urine was recovered over the first 12 h post-dosing; in faeces, the majority of the dose was recovered in the 12 to 24 h collection interval with significant amounts still being excreted over 24 to 48 h. Greater than 95% of the total amount recovered was generally collected in the excreta in the first 48 h following dosing. In order to determine what fraction of the dose that was recovered in the faeces was absorbed prior to excretion, and due to the complexity of the initial HPLC radio profiles of faecal homogenate extracts, four BDC animals per labelled test substance were administered a single oral dose at 100 mg/kg. In the BDC rats, approximately 60% of the [PH-U-14C]-test substance dose was recovered in the bile, 21% in the urine, and 3.3% in the faeces over 48 h post-dosing, indicating that >95% of the recovered dose was absorbed. The [TH-2-14C]-test substance dose was primarily recovered in the urine (45%) and the bile (32.2%), followed by the faeces (11%).

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

LC/MS/MS analysis confirmed that there was no intact [PH-U-14C]-test substance or [TH-2-14C]-test substance in urine samples. [TH-2-14C]-test substance and [PH-U-14C]-test substance were each extensively metabolized to approximately 29 and 30 components, respectively, and most metabolites represented <5% of the radioactivity in any individual sample, and <1% of the dose administered overall. As a percent of dose, benzamidine, identified by LC/MS and retention time comparison to an authentic standard, was the most abundant metabolite in urine (4.1% to 12.6%) followed by M39 identified as hydroxy test substance glucuronide (1.2% to 4.6%), and hippuric acid (1.0% to 2.6%) after [PH-U-14C]-test substance dosing. As a percent of dose, thenoylglycine (0.7% to 6.2%), and glucuronide conjugate M39 (3.6% to 4.7%) were the most abundant metabolites in urine after [TH-2-14C]-test substance dosing. Benzamidine also was the most abundant metabolite in the faecal homogenates from all groups dosed with [PH-U-14C]-test substance constituting 9.1% to 26.2% of the administered dose.

Smaller poorly resolved peaks were present in all faecal chromatograms following the administration of either label in the same regions as were identified in the urine profiles but they were minor contributors to the total overall radioactivity. Bile radiochromatograms indicated one primary peak following a single oral dose of [PH-U-14C]-test substance or [TH-2-14C]-test substance to male rats. Further investigation by LC/MS/MS identified/characterized up to 29 [PH-U-14C]-test substance metabolites and 20 [TH-2-14C]-test substance metabolites in bile. Mass spectral data showed that the structure of the most abundant test substance metabolite (M39, 23% to 27% of the administered dose) is Hydroxyl test substance Glucuronide, a metabolite in which test substance has been hydroxylated at the 5-position of the thiophene ring and glucuronidated.

Any other information on results incl. tables

Pathways of metabolism:

The major proposed pathways for metabolism of test substance in rats are:

1) Reductive cleavage of the N-O bond of the oxadiazole ring leading to test substance Iminoamide, a transient metabolite that is not observed as a free metabolite in any matrix. The iminoamide metabolite is hydrolyzed (almost certainly enzyme-mediated) to benzamidine, the major urine and fecal metabolite, which is also further hydrolyzed to benzoic acid (eliminated in urine as the glycine conjugate, hippuric acid). Hydrolysis of the iminoamide also gives 2-thiophenecarboxylic acid (eliminated as 2-thenoylglycine).

2) Hydroxylation of the thiophene ring (primarily at the 5-position of the ring, adjacent to the sulfur atom) and conjugation as the glucuronide (major) or sulfate.

Additional proposed pathways of metabolism were: dihydroxylation of the thiophene ring and conjugation with glucuronic acid, sulfate or glucose; glutathione substitution on the thiophene ring and catabolism to the mercapturate; oxidative ring-opening of the thiophene ring; and oxidation of the sulfur atom of the thiophene ring forming the thiophene-S-oxide. For all groups (except the 3 mg/kg [PH-U-14C]-test substance females), the mean concentration in tissues at 168 h post-dosing following a 3 mg/kg dose was generally <0.1 μg/g with the exception of the adrenal glands (mean concentration across all groups 0.3 μg/g to 0.73 μg/g), the kidneys (0.14 μg/g to 0.32 μg/g), the liver (0.08 μg/g to 0.16 μg/g), and the thyroid (0.07 μg/g to 0.23 μg/g). As a percent of dose, the greatest recovery was in the liver (up to 0.23% of the dose).

The mean amount recovered in the remaining carcass was 0.43% to 0.73% of the administered dose. Although the average recovery of the administered dose was only 47.7% for female rats dosed at 3 mg/kg with [PH-U-14C]-test substance, tissue distribution results were similar to those of the other groups. Mean tissue concentrations for the females were ≤0.02 μg/g in all tissues except adrenal glands (0.11 μg/g), kidneys (0.11 μg/g), liver (0.06 μg/g) and the thyroid (0.03 μg/g). There was no apparent trend in tissue distribution at 168 h post-dosing related to the position of the radiolabel, the sex of the rats, or single versus repeated dose administration.

The concentration in tissues at 168 h post-dosing following a 100 mg/kg dose was highest for the adrenal gland (14 μg/g to 16 μg/g), followed by the kidneys (4.6 μg/g to 7.0 μg/g), liver (3.0 μg/g to 3.6 μg/g), and the thyroid (2.8 μg/g to 3.6 μg/g). All other tissues had mean concentrations ranging from 0.07 μg/g to 1.7 μg/g. On a dose-adjusted basis, tissue concentrations were comparable between the 3 mg/kg and 100 mg/kg dose groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
Under the test conditions, test substance was well absorbed following oral administration to rats. It was extensively metabolized and rapidly excreted, primarily via the bile and faeces, with little tissue retention. Major routes of metabolism of test substance in rats were oxidation (hydroxylation) of the thiophene ring, followed by conjugation primarily with glucuronic acid, and reductive cleavage and subsequent hydrolysis of the oxadiazole ring.

Executive summary:

A study was performed to determine the pharmacokinetics, routes of elimination, mass balance, tissue distribution, and metabolite profiles of [14C] labeled residues after oral or intravenous administration of [phenyl (PH)-UL-14C] labeled test substance or [thiophene (TH)-2 -14C] labeled test substance at dose 3 and 100 mg/kg bw to rats, according to OECD Guideline 417 and OPPTS 870.7485, in compliance with GLP. This study was conducted in multiple phases: a pilot phase, a pharmacokinetic phase, a disposition and metabolite identification phase, and a quantitative whole body autoradiography (QWBA) phase. Also, major metabolites of these compounds were quantified and identified or characterized to the extent possible using LC/MS/MS. Test substance was well absorbed following oral administration to rats. It was extensively metabolized and rapidly excreted, primarily via the bile and faeces, with little tissue retention. There may have been a slight increase in relative plasma levels of test substance and/or its metabolites after dosing at 100 mg/kg bw vs. 3 mg/kg bw but there were no major differences in excretion or metabolism due to route of administration, dose level, sex of the animals, or single vs. repeat dosing. Major routes of metabolism of test substance in rats were oxidation (hydroxylation) of the thiophene ring, followed by conjugation primarily with glucuronic acid, and reductive cleavage and subsequent hydrolysis of the oxadiazole ring (Thomas J, 2014).