1-[5-(2,6-difluorophenyl)-4,5-dihydro-1,2-oxazol-3-yl]ethanone

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Objective of study:

absorption

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

- Purity: 99.9%

Radiolabelling:

no

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 340 - 372 g
- Fasting period before study: Animals for the oral and intravenous administration experiments were fasted (approximately 16 ± 2 hours) before dosing with test substance. Animals for the repeat dose 14-day oral administration experiment were not fasted before dosing with the test substance. Food was returned approximately 2 hours post dose.
- Housing: During the pretest period, animals were group housed (non-cannulated) or housed individually (cannulated) in solid bottom caging with Bed-O-Cob® and Nestlets. For the in-life phase, animals were housed individually in solid-bottom caging with Bed-O-Cob® and Nestlets. The exception was for groups 1 and 2 that were housed in pairs on day 0 before being housed individually starting on day 1.
- Food: All animals were fed PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum, except when fasted.
- Water: tap water ad libitum
- Acclimation period: quarantined for at least 5 days with the exception of the cannulated rats, which were quarantined for at least 3 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26ºC
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

other: single oral gavage, intravenous, or 14-day repeated oral gavage

Vehicle:

other: oral gavage doses were prepared with 0.1% Tween 80 in 0.5% methylcellulose; intravenous dose was prepared with 5% ethanol, 5% alkamuls, and 90% physiological saline

Details on exposure:

PREPARATION OF TEST SUBSTANCE FORMULATION:
- Preparation frequency: once
- Preparation details: The test item was mixed with 0.5% methylcellulose with 0.1% Tween 80 for oral gavage administration or 5% ethanol, 5% alkamuls (a surfactant), and 90% physiological saline for intravenous administration. The formulation for oral administration was a homogenous suspension and the intravenous formulation was visibly observed to be a clear solution. Furthermore, the intravenous dose was sterile filtered through a 0.22 μm filter.
- Adjusted for purity: no

Duration and frequency of treatment / exposure:

Single oral gavage administration
14-day repeated oral gavage administration
Single intravenous administration

Doses / concentrations

Remarks:

Doses / Concentrations:
Single oral gavage: 100 (Group 1) or 1000 mg/kg bw (Group 2)
14-day repeated oral gavage: 100 mg/kg bw (Group 4)
Single intravenous: 5 mg/kg bw (Group 3)

No. of animals per sex per dose / concentration:

4 per dose level

Control animals:

no

Details on study design:

- Dose selection rationale:
The high dose was set at 1000 mg/kg bw, which is the maximum dose recommended in OECD, Section 4, (Part 417): Toxicokinetics, Guidelines for the Testing of Chemicals (2010). The low dose was set at 100 mg/kg bw, which is below a dose that did not cause clinical signs in an acute oral toxicity study in rats. For the intravenous administration experiment, a dose of 5 mg/kg bw was chosen, which is 1/20 of the low dose for the oral gavage experiment. For the repeated dose 14-day oral gavage experiment, the dose was set to 100 mg/kg bw, which is equal to the low dose for the oral gavage experiment.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood from tail vein
- Time and frequency of sampling:
Oral gavage - 15 and 30 min, as well as at 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours
Intravenous administration - 2, 5, 15, and 30 min, as well as at 1, 2, 6, 12, 24, 48, 72, 96, 120, 144, and 168 hours
- Other: Blood was processed into plasma, which was stored frozen at ≤ -10°C until it was analysed
- From how many animals: samples pooled across the 4 animals
- Method type(s) for identification: HPLC-MS
- Limits of detection and quantification: The LOD and LOQ for the test item were 12.0 and 40.0 ng/mL, respectively.

Statistics:

Group data was represented as a mean ± SD as appropriate. Tables and appendices presented in the report were computer generated and values were rounded appropriately for inclusion in the report. Consequently, the application of manual calculation to report values may have, in some instances, yielded a minor variation. These occurrences should not be construed as adversely affecting the integrity or interpretation of the data.

Results and discussion

Main ADME results

Type:

absorption

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The pharmacokinetics of the test item showed both rapid absorption and elimination. Metabolism of the absorbed dose was extensive and characterized by 23 tentatively identified components in plasma. The results from these experiments (single oral gavage, intravenous, and 14-day repeated oral gavage administration) indicate that the test item is metabolized rapidly and extensively and is not likely to accumulate following multiple dosing.

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 59.3 (±4.1) ng/mL single low dose oral

Toxicokinetic parameters 2

Key result

Test no.:

#2

Toxicokinetic parameters:

Cmax: 205 (±42) ng/mL single high dose oral

Toxicokinetic parameters 3

Key result

Test no.:

#3

Toxicokinetic parameters:

Tmax: 1.2 (±0.8) hours single low dose oral

Toxicokinetic parameters 4

Key result

Test no.:

#4

Toxicokinetic parameters:

Tmax: 0.63 (±0.25) hours single high dose oral

Toxicokinetic parameters 5

Key result

Test no.:

#5

Toxicokinetic parameters:

AUC: 942 (±466) hr x ng/mL single high dose oral

Toxicokinetic parameters 6

Key result

Test no.:

#6

Toxicokinetic parameters:

half-life 1st: 2.0 (±0.4) hours single high dose oral

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Double-bond reduction, hydroxylation, sulfation – Isomer I
(+2H, +O, +SO3)
Double-bond reduction, hydroxylation, sulfation – Isomer II
(+2H, +O, +SO3)
Double-bond reduction, hydroxylation, glucuronidation – Isomer I
(+2H, +O, +C6H8O6, +SO3)
Double-bond reduction, hydroxylation, glucuronidation – Isomer II (+2H, +O, +C6H8O6,+SO3)
Double-bond reduction, hydroxylation, glucuronidation – Isomer III (+2H, +O, +C6H8O6,+SO3)
Double-bond reduction, sulfation – Isomer I (+2H, +SO3)
Hydroxylation, glucuronidation – Isomer I (+O, +C6H8O6)
Hydroxylation, glucuronidation– Isomer II (+O, +C6H8O6)
Hydroxylation, glucuronidation – Isomer III (+O, +C6H8O6)
Double-bond reduction, glucuronidation – Isomer I
Double-bond reduction, glucuronidation – Isomer II
(+2H, +C6H8O6)
Di-hydroxylation – Isomer I (+2O)
Glucuronidation, defluorination and hydroxylation, iso-ring opening, oxidation of C=N to C=O and then reduction to alcohol – Isomer I
(+C6H8O6, -F, +O, +H, -N, +O, +3H)
Glucuronidation, defluorination and hydroxylation, iso-ring opening, oxidation of C=N to C=O and then reduction to alcohol – Isomer II
(+C6H8O6, -F, +O, +H, -N, +O, +3H)
unidentified metabolite (M+H= C10H9FNO5) – Isomer I
unidentified metabolite (M+H= C10H9FNO5) – Isomer II
N-Glucuronidation – Isomer I (+C6H8O6)
Trace
N-Glucuronidation – Isomer II (+C6H8O6)
Formation of carbonyl, and hydroxylation (+2O, -2H)
Double-bond reduction, hydroxylation – Isomer I (+2H, +O)
Double-bond reduction, hydroxylation – Isomer II (+2H, +O)
Defluorination and hydroxylation, iso-ring opening, oxidation of C=N to C=O and then reduction to alcohol (-F, +O, +H, -N, +O, +3H)
Double-bond reduction (+2H)

Any other information on results incl. tables

There were no clinical signs observed for groups 1, 2, and 3. For group 4, one rat had diarrhoea on test day 9. All rats appeared normal upon subsequent observation.

Pharmacokinetics of the test item in plasma following single oral gavage administration showed both rapid absorption and elimination. Mean peak plasma concentrations (Cmax) at the low and high dose were 59.3 (±4.1) and 205 (±42) ng/mL, respectively. Mean Tmax values were 1.2 (±0.8) and 0.63 (±0.25) hours at the low and high dose respectively. Concentrations of the test item were (above the LOQ) in plasma for up to 2 hours after dose administration in the low-dose group and 24 hours in the high-dose group. In some, but not all of the rats in the high dose group, plasma concentrations of the test item fell below the LOQ at the 8- and/or 12-hour time point before increasing to above the LOQ at 24 hours.

The mean plasma terminal elimination half-life value and area-under-the-curve (AUC) for the single oral gavage high dose group was 2.0 (±0.4) hours and 942 (±466) hr x ng/mL, respectively. The half-life and AUC could not be determined for the single oral gavage low dose group because it lacked enough time points with quantifiable concentrations of the test item in plasma.

Plasma concentrations of the test item were not quantifiable at any of the measured time points after the intravenous administration and after the repeated dose 14-day oral gavage administration experiments. Therefore, the pharmacokinetic parameters terminal half-life, AUC, Cmax, Tmax, volume of distribution, oral bioavailability, and clearance were not determined for these groups. However, in order to determine the fate of the test item, plasma samples at the 1-hour time point from each dose group were submitted for high resolution/high mass accuracy mass spectrometry to tentatively determine structures of the metabolites.  The test item metabolized readily to 23 tentatively identified components. Qualitatively, metabolism in the rat was similar among the different dose groups.

In summary, the pharmacokinetics of the test item showed both rapid absorption and elimination. Metabolism of the absorbed dose was extensive and characterized by 23 tentatively identified components in plasma. The results from these experiments (single oral gavage, intravenous, and 14-day repeated oral gavage administration) indicate that the test item metabolized rapidly and extensively and is not likely to accumulate following multiple dosing.

Applicant's summary and conclusion

Conclusions:

The pharmacokinetics of the test item showed both rapid absorption and elimination. Metabolism of the absorbed dose was extensive and characterized by 23 tentatively identified components in plasma. The results from these experiments (single oral gavage, intravenous, and 14-day repeated oral gavage administration) indicate that the test item metabolized rapidly and extensively and is not likely to accumulate following multiple dosing.

Executive summary:

The toxicokinetics of the test item was studied in male Sprague-Dawley rats. Experiments were performed to understand plasma kinetics following single oral gavage, intravenous, or 14-day repeated oral gavage administration. Four male rats were administered the test item at 100 or 1000 mg/kg bw by single oral gavage. Another four male rats were administered the test item at 100 mg/kg bw daily by gavage for 14 days. Following the last administered dose, blood was collected via the tail vein at 15 and 30 min, as well as at 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours.

There were no clinical signs observed for groups 1, 2, and 3. For group 4, one rat had diarrhoea on test day 9. All rats appeared normal upon subsequent observation. Pharmacokinetics of the test item in plasma following single oral gavage administration showed both rapid absorption and elimination.  Mean peak plasma concentrations (Cmax) at the low and high dose were 59.3 (±4.1) and 205 (±42) ng/mL, respectively. Mean Tmax values were 1.2 (±0.8) and 0.63 (±0.25) hours at the low and high dose respectively.  Concentrations of the test item were (above the LOQ) in plasma for up to 2 hours after dose administration in the low-dose group and 24 hours in the high-dose group. In some, but not all of the rats in the high dose group, plasma concentrations of the test item fell below the LOQ at the 8- and/or 12-hour time point before increasing to above the LOQ at 24 hours.

The mean plasma terminal elimination half-life value and area-under-the-curve (AUC) for the single oral gavage high dose group was 2.0 (±0.4) hours and 942 (±466) hr x ng/mL, respectively.  The half-life and AUC could not be determined for the single oral gavage low dose group because it lacked enough time points with quantifiable concentrations of the test item in plasma. Plasma concentrations of the test item were not quantifiable at any of the measured time points after the intravenous administration and after the repeated dose 14-day oral gavage administration experiments.  Therefore, the pharmacokinetic parameters terminal half-life, AUC, Cmax, Tmax, volume of distribution, oral bioavailability, and clearance were not determined for these groups. However, in order to determine the fate of the test item, plasma samples at the 1-hour time point from each dose group were submitted for high resolution/high mass accuracy mass spectrometry to tentatively determine structures of the metabolites.  The test item metabolized readily to 23 tentatively identified components.  Qualitatively, metabolism in the rat was similar among the different dose groups.

In summary, the pharmacokinetics of the test item showed both rapid absorption and elimination.  Metabolism of the absorbed dose was extensive and characterized by 23 tentatively identified components in plasma.  The results from these experiments (single oral gavage, intravenous, and 14-day repeated oral gavage administration) indicate that the test item metabolized rapidly and extensively and is not likely to accumulate following multiple dosing.