3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione;famoxadone (ISO)

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: E91/414/EEC [Annex III, Point 7]

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Pesticide Assessment Guidelines Subdivision F, Section 85-2

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

Substance name: [14C-U-phenoxyphenyl]DPX-JE874
Radiochemical purity: 98%
Specific activity: 61.8 µCi/mg

Substance name: DPX-JE874
Lot #: 352DE1 REF92
Purity: 99.6%

Radiolabelling:

yes

Species:

rat

Strain:

other: CRl:CD(SD)BR

Sex:

female

Type of coverage:

semiocclusive

Vehicle:

other: Acetone or deionized water

Duration of exposure:

8, 24, 48, 144 h

Doses:

10 (formulation concentrate), 0.75 (aqueous solution) mg/animal

No. of animals per group:

4

Control animals:

no

Signs and symptoms of toxicity:

no effects

Remarks:

No overt pharmacological or toxicological signs were observed in the test animals which could have been attributed to the administration of [14C]test substance.

Total recovery:

An acceptable material balance (ca 87% of the dermally applied radioactivity, range 84 to 90% for all 4 sampling groups) was attained. The majority of the applied radioactivity was recovered in the back washes (>64% of the dermally applied radioactivity) at the 8 and 24 hours washing intervals, and from the washings of the bandages and protective appliances (5 to 18%). At the 24 h washing interval, only ca 5% of the applied radioactivity remained at the application site after washing. When test animals were sacrificed at the 72 and 168 h sampling intervals, only low levels of radioactivity (0.01 to 0.9%) were recovered from the second back wash and solubilised skin. Low levels of radioactivity were recovered in the excreta, surrounding skin area and remaining carcass. Negligible levels of 14C-residue were observed in the blood with values at, or close to the limit of detection. Approximately 4% of the applied radioactivity (mainly in the faeces) was recovered in the excreta, 168 h after treatment. This distribution data showed the limited in vivo dermal penetration of [14C]test substance after dermal application of DPX-MC444-11 formulation concentrate to female laboratory rats.

The overall distribution profile of the administered [14C]test substance radioactivity in this treatment group after exposure to a dilute aqueous solution of DPX-MC444-11 was similar to that observed with the undiluted concentrate formulation. An acceptable material balance (>85%) was attained. The majority of the applied radioactivity was recovered in the application site skin washes, bandages and protective appliance. Negligible levels of 14C-residue were observed in the blood with values at, or close to the limit of detection. Approximately 11% of the applied radioactivity remained at the application site after the 24 h washing interval, and was subsequently dissipated at the 72 and 168 h sacrifice intervals. Approximately 7% of the applied radioactivity was recovered in the combined excreta (mainly faeces), application skin site and remaining carcass, which further confirmed the limited dermal penetration of [14C]test substance in the diluted aqueous DPX-MC444-11 solution.

Key result

Time point:

8 h

Dose:

10 mg [14C]DPX-JE874 (formulation concentrate)

Parameter:

percentage

Absorption:

4.31 %

Key result

Time point:

8 h

Dose:

0.75 mg [14C]DPX-JE874 (aqueous solution)

Parameter:

percentage

Absorption:

5.37 %

Key result

Time point:

24 h

Dose:

10 mg [14C]DPX-JE874 (formulation concentrate)

Parameter:

percentage

Absorption:

5.86 %

Key result

Time point:

24 h

Dose:

0.75 mg [14C]DPX-JE874 (aqueous solution)

Parameter:

percentage

Absorption:

13.77 %

Time point:

72 h

Dose:

10 mg [14C]DPX-JE874 (formulation concentrate)

Parameter:

percentage

Absorption:

3.07 %

Time point:

72 h

Dose:

0.75 mg [14C]DPX-JE874 (aqueous solution)

Parameter:

percentage

Absorption:

9.27 %

Time point:

168 h

Dose:

10 mg [14C]DPX-JE874 (formulation concentrate)

Parameter:

percentage

Absorption:

4.93 %

Time point:

168 h

Dose:

0.75 mg [14C]DPX-JE874 (aqueous solution)

Parameter:

percentage

Absorption:

10.1 %

Conclusions:

The maximum dermal absorption occurred within 24 hours after exposure. An approximately 2-fold difference in the percent penetration was observed in the aqueous test solution.

Executive summary:

This study was designed to investigate the in vivo dermal penetration of [14C]test substance (and/or its metabolites) in female laboratory rats, following a single dermal administration of [14C]DPX-MC444-11 formulation at two dose levels: as the undiluted emulsifiable concentrate and as an aqueous dilution of the concentrate equivalent to approximately ten times that of the field application solution.

Overall, limited dermal penetration of test substance was observed. The majority of administered radioactivity was removed at 8 h or 24 h by washing of the application site. Maximal dermal absorption occurred within 24 h after the initial application. Dermal absorption (expressed as the percent of the applied radioactivity) for the aqueous solution was approximately twice that for the formulation concentrate.

Negligible amounts (<1%) of the applied radioactivity was recovered in the urine. Faeces accounted for approximately 6% of the applied radioactivity. The majority of the applied radioactivity was removed in the skin wash.

Analysis of the skin washes, by HPLC, following 24 h exposure showed the presence of a single radioactive component with a retention time consistent with that of the test substance. Excreted radioactivity was largely eliminated as test substance in the faeces with some IN-KZ007 also present.

In conclusion, following a single dermal administration of [14C]test substance, limited dermal penetration was observed, with the majority of excreted radioactivity eliminated in the faeces. Washing the application site effectively removed the majority of applied radioactivity.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

GLP compliance:

yes

Specific details on test material used for the study:

Substance name: [14C-PA]DPX-JE874
Purity: Not reported

Radiolabelling:

yes

Species:

dog

Strain:

Beagle

Sex:

male

Route of administration:

oral: unspecified

Vehicle:

not specified

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

15 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

3

Control animals:

no

Type:

metabolism

Results:

A single component with a retention time similar to DPX-JE874 was strongly predominant in the HPLC profiles of fat extracts and the identification was confirmed by mass spectroscopy. A minor region of radioactivity, which included KZ007, was also observed

Type:

metabolism

Results:

DPX-JE874 and KZ007 were identified by mass spectroscopy as prominent components in liver extracts.

Type:

metabolism

Results:

RBC: Mass spectroscopic analysis confirmed that parent DPX-JE874 and metabolites KZ007, ML815 and JL856 were present.

Type:

metabolism

Results:

Plasma: DPX-JE874, KZ007, ML815 and JL856 were identified by mass spectroscopy. However, the amount of DPX-JE874 was lower in plasma, suggesting that the partition equilibrium favored distribution into red blood cells.

Details on excretion:

A complex pattern of radiolabelled metabolites were observed in urine and faeces. Urine contained mainly polar components, none of which could be identified with reference to the known metabolites of the phenylamino moiety of DPX-JE874; there was no evidence to suggest that glucuronide or sulphate conjugates were present. Faeces extracts contained mainly DPX-JE874 at early collection times, but more metabolites were formed at later times, which included ML815, KZ534, KZ532, KZ007 and JL856.

Metabolites identified:

yes

Details on metabolites:

Up to twelve regions of radioactivity were assigned in the HPLC profiles of red blood cell extracts. The most polar region was predominant and a region with retention time similar to DPX-JE874 was also prominent. Mass spectroscopic analysis confirmed that parent DPX-JE874 and metabolites KZ007, ML815 and JL856 were present.

The HPLC profiles of plasma extracts contained eleven regions of radioactivity and, as for red blood cells, DPX-JE874, KZ007, ML815 and JL856 were identified by mass spectroscopy. However, the amount of DPX-JE874 was lower in plasma, suggesting that the partition equilibrium favored distribution into red blood cells.

Up to eight regions of radioactivity were observed in the HPLC profiles of liver extracts, of which the major proportion of radioactivity eluted with a retention time >25 min. DPX-JE874 and KZ007 were identified by mass spectroscopy as prominent components in liver extracts.

A single component with a retention time similar to DPX-JE874 was strongly predominant in the HPLC profiles of fat extracts and the identification was confirmed by mass spectroscopy. A minor region of radioactivity, which included KZ007, was also observed in the extract from one animal.

Conclusions:

Following a single oral dose of test substance to dogs, the circulating metabolites in plasma which were quantified included parent DPX-JE874 and the metabolites KZ007, JL856 and ML815. These were also present in red blood cells. Extracts of liver and fat were shown to contain predominantly DPX-JE874 with lesser amounts of KZ007. No further metabolites were noted in fat, but liver contained several other unidentified metabolites.

Executive summary:

The number and nature of radiolabelled metabolites, present in samples of red blood cells, plasma, liver and fat taken 2 h after a single oral dose of [14C-PA]test substance, labelled in the phenylamino moiety, to the dog at a nominal dose level of 15 mg/kg body weight, have been evaluated. In addition, excreta (faeces and urine) have been examined for radiolabelled metabolites at intervals over a 96 h post-dose period. Furthermore, four plasma components, namely DPX-JE874, ML813, KZ007 and JL856, have been quantified in samples taken from separate animals at frequent intervals up to 96 h post-dose.

Up to twelve regions of radioactivity were assigned in the HPLC profiles of red blood cell extracts. The most polar region was predominant and a region with retention time similar to DPX-JE874 was also prominent. Mass spectroscopic analysis confirmed that parent DPX-JE874 and metabolites KZ007, ML815 and JL856 were present.

The HPLC profiles of plasma extracts contained eleven regions of radioactivity and, as for red blood cells, DPX-JE874, KZ007, ML815 and JL856 were identified by mass spectroscopy. However, the amount of DPX-JE874 was lower in plasma, suggesting that the partition equilibrium favored distribution into red blood cells.

Up to eight regions of radioactivity were observed in the HPLC profiles of liver extracts, of which the major proportion of radioactivity eluted with a retention time >25 min. DPX-JE874 and KZ007 were identified by mass spectroscopy as prominent components in liver extracts.

A single component with a retention time similar to DPX-JE874 was strongly predominant in the HPLC profiles of fat extracts and the identification was confirmed by mass spectroscopy. A minor region of radioactivity, which included KZ007, was also observed in the extract from one animal.

It was not possible to extract radioactivity from aqueous humor due to the very low concentration of radioactivity and small sample size.

A complex pattern of metabolites was detected in HPLC profiles of urine samples (pooled by collection time), with up to eight radioactive regions assigned to each chromatogram. The nature of each region remained unchanged generally, although the proportion of radioactivity associated with each region varied with collection time. All regions eluted with a retention time shorter than that for test substance reference material. None gave similar mass spectra to known metabolites derived from the phenylamino moiety of test substance. No structure, eg acetanilide, acetamidophenol, aniline, aminophenol etc, could be assigned based on mass spectroscopic characteristics. Enzyme hydrolysis suggested that glucuronide and sulphate conjugates were absent.

A minor polar region and five less polar (i.e., retention time >25 min) regions were assigned in the HPLC profiles of pooled faeces extracts. One of the less polar regions, with a retention time similar to parent DPX-JE874, was predominant in profiles at early collection times. At later times, the majority of radioactivity was associated with metabolites which were considered to have retained the non-polar characteristics of the parent molecule. Evidence was obtained by mass spectrometry to confirm the presence of DPX-JE874 and metabolites KZ007, ML815, JL856, KZ532 and KZ534 in faeces extracts.

Plasma samples from three separate dogs, collected up to 96 h after a single oral dose of [14C-PA]test substance, were assayed for radioactivity and for DPX-JE874, KZ007, JL856 and ML815 using a validated method. Bimodal absorption profiles were observed for radioactivity and this profile was very pronounced in one animal. Similar profiles were observed for KZ007 which was the component detected at the highest concentration. Furthermore, most analytes showed some degree of recycling which precluded pharmacokinetic analysis. Visual inspection of the concentration / time profiles showed that the analytes quantified accounted for only a small proportion of the overall radioactivity.

In conclusion, following a single oral dose of test substance to dogs, the circulating metabolites in plasma which were quantified included parent DPX-JE874 and the metabolites KZ007, JL856 and ML815. These were also present in red blood cells. Some recycling of these metabolites was evident, leading to multimodal concentration / time profiles. Other metabolites were present in plasma but their identity could not be elucidated. Extracts of liver and fat were shown to contain predominantly DPX-JE874 with lesser amounts of KZ007. No further metabolites were noted in fat, but liver contained several other unidentified metabolites. A complex pattern of radiolabelled metabolites were observed in urine and faeces. Urine contained mainly polar components, none of which could be identified with reference to the known metabolites of the phenylamino moiety of DPX-JE874; there was no evidence to suggest that glucuronide or sulphate conjugates were present. Faeces extracts contained mainly DPX-JE874 at early collection times, but more metabolites were formed at later times, which included ML815, KZ534, KZ532, KZ007 and JL856.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

distribution

excretion

Qualifier:

no guideline followed

Principles of method if other than guideline:

The absorption, distribution, and elimination of radioactivity were studied in male beagle dogs dosed with [14C-PA]test substance. The animals were divided into three groups: 3, 3 and 1 animal for Group A, Group B and Group C, respectively. Group A and Group В dogs were dosed once orally by gavage with [14C-PA]test substance at 15 mg/kg, and the Group C dog was dosed with carrier solution only. Urine and feces were collected from Group A and Group C animals at specified time intervals. Group A animals were sacrificed 4 days after administration of the radiolabeled dose. Selected tissues, excreta samples, blood and plasma were analyzed for radioactivity. Group В animals were sacrificed at the time of the Сmax of the Group A plasma concentrations. Group C animals were sacrificed 4 days after administration of the carrier solution.

GLP compliance:

yes

Specific details on test material used for the study:

Radioactive test substance
[14C-PA]DPX-JE874
Lot #: 3048-238
Radiochemical purity: 98.7%
Specific activity: 58.529 mCi/mmole

Non-Radioactive test substance
DPX-JE874
Lot # JE874-92
Purity: 99.6%

Radiolabelling:

yes

Species:

dog

Strain:

Beagle

Details on species / strain selection:

This species represents a preferred nonrodent species for developing data on the absorption, distribution, metabolism, and excretion of a test chemical to aid in the evaluation of test results from toxicology studies and in the extrapolation of data from animals to humans.

Sex:

male

Route of administration:

oral: gavage

Vehicle:

other: 0.5% polysorbate 80 (Tween 80) in 1% sodium carboxymethyl cellulose (CMC)

Duration and frequency of treatment / exposure:

Each dog received a single oral dose administered via gavage, followed by approximately 10 mL of water to flush the dosing tube

Dose / conc.:

15 mg/kg bw/day

No. of animals per sex per dose / concentration:

Group A & B: 03 males each
Group C (control): 01 male

Control animals:

yes, concurrent vehicle

Type:

absorption

Results:

The test material was rapidly absorbed

Type:

distribution

Results:

The concentrations of radioactive residues in the tissues were highest in the fat and the liver for both Group A and Group В dogs.

Type:

excretion

Results:

Overall, the mean recovery of radioactivity from tissues was 0.45%, with a range of 0.23% to 0.86%.

Details on absorption:

The test material was rapidly absorbed. The highest mean radioactivity concentration in the plasma during the pharmacokinetics phase (Group А Сmaх) was 1.53 ppm [14C-PA]test substance µg equivalents/g) and the Tmax occurred 2 hours after the dose. The highest mean radioactivity concentration in RBCs measured during the first 12 hours of the pharmacokinetic phase (Group A) was 0.626 ppm and occurred 4 hours postdose. The terminal half-life (t1/2) and area under the curve (AUC0-∞) ranged from 67 to 75 hours and 98 to 109 µg/g.hour for the plasma and 146 to 159 hours and 125 to 135 µg/g.hour for the RBCs, respectively.

Details on distribution in tissues:

The highest mean concentrations from Group A were detected in the liver (1.34 ppm) and mesenteric fat (0.945 ppm). As was observed in the plasma and RBCs, the concentrations of radioactivity in the liver and fat from Animal No. H09704 were significantly higher than, the concentrations of similar samples from the other two dogs (again, approximately three times higher). The reason for the higher concentrations of radioactivity in plasma, RBCs, and tissues was not determined.
The recovery of radioactivity in the excreta from the animal in question was midrange to the other dogs (84.4% of dose administered versus 65.8% and 86.0% for the other two dogs, respectively). Because the dog was in the top cage, contamination from another cage was not possible. There were no observations made of unusual events for this animal. It is possible that following the 12 hour blood collection the dog ingested some of its own feces, resulting in the increase in plasma, RBC and tissue concentrations (essentially a redose). It is also possible that the dog vomited and aspirated the vomitus into the lungs. The exposure of the test material in the stomach contents to the thin-membrane, blood-rich lung tissue could have resulted in the increased uptake of the test material into the bloodstream. The ingestion of feces or aspiration of vomit could explain why there was no effect on the overall recovery of radioactivity but that higher recoveries of radioactivity in the urine/feces collections were shifted to later times.
There were no radioactive residues detected in any Group C (control) samples.
The Group В dogs were dosed with test material at 15 mg/kg and were sacrificed at the Cmax from the Group A plasma levels (2 hours after dosing). Highest mean residue concentrations were seen in the liver (4.45 ppm), mesenteric fat (2.80 ppm), plasma (0.999 ppm), and RBCs (0.413 ppm). The residues in the aqueous humor, eye, and eye remainder were 0.061 ppm, 0.106 ppm, and 0.131 ppm, respectively. Urine and feces were not collected.

Details on excretion:

The total mean recovery of radioactivity in excreta from dogs in Group A was 78.8%, with 7.67% eliminated in urine and 70.3% eliminated in feces (the remainder in cage washes and wipes).
The elimination rate of radioactivity was fairly slow from plasma and tissues. The majority (approximately 65.0%) of the radioactivity was eliminated in the urine and feces within 24 hours after dosing but high levels of radioactivity remained in the plasma and RBCs through 96 hours postdose (Group A).

Test no.:

#1

Toxicokinetic parameters:

Tmax: 2 hours (plasma)

Test no.:

#1

Toxicokinetic parameters:

Cmax: 1.53 ppm

Test no.:

#1

Toxicokinetic parameters:

AUC: 98 to 109 µg/g.hour for the plasma and 125 to 135 µg/g.hour for the RBCs

Test no.:

#1

Toxicokinetic parameters:

other: t1/2: 67 to 75 hours for plasma and 146 to 159 hours for RBC's

Conclusions:

Male beagle dogs that were treated with [14C-PA]test substance at a dose level of 15 mg/kg excreted most of the radioactivity within 96 hours after dosing. Urine, feces, cage washes, and cage wipes accounted for 78.8%, with a range of 65.8% to 86.0% of the administered radioactivity to Group A. Overall, the mean recovery of radioactivity from tissues was 0.45%, with a range of 0.23% to 0.86%.

Executive summary:

The absorption, distribution, and elimination of radioactivity were studied in male beagle dogs dosed with [14C-PA]test substance. The animals were divided into three groups: 3, 3 and 1 animal for Group A, Group B and Group C, respectively. Group A and Group В dogs were dosed once orally by gavage with [14C-PA]test substance at 15 mg/kg, and the Group C dog was dosed with carrier solution only. Urine and feces were collected from Group A and Group C animals at specified time intervals. Group A animals were sacrificed 4 days after administration of the radiolabeled dose. Selected tissues, excreta samples, blood and plasma were analyzed for radioactivity. Group В animals were sacrificed at the time of the Сmax of the Group A plasma concentrations. Group C animals were sacrificed 4 days after administration of the carrier solution.

The total mean recovery of radioactivity in excreta from dogs in Group A was 78.8%, with 7.67% eliminated in urine and 70.3% eliminated in feces (the remainder in cage washes and wipes).

The elimination rate of radioactivity was fairly slow from plasma and tissues. The majority (approximately 65.0%) of the radioactivity was eliminated in the urine and feces within 24 hours after dosing but high levels of radioactivity remained in the plasma and RBCs through 96 hours postdose (Group A).

The highest mean radioactivity concentration in the plasma during the pharmacokinetics phase (Group А Сmaх) was 1.53 ppm [14C-PA]test substance µg equivalents/g) and the Tmax occurred 2 hours after the dose. The highest mean radioactivity concentration in RBCs measured during the first 12 hours of the pharmacokinetic phase (Group A) was 0.626 ppm and occurred 4 hours postdose. The terminal half-life (t1/2) and area under the curve (AUC0-∞) ranged from 67 to 75 hours and 98 to 109 µg/g.hour for the plasma and 146 to 159 hours and 125 to 135 µg/g.hour for the RBCs, respectively.

One of the three dogs in Group A (Animal No. H09704) had a plasma and RBC profile that was similar to the other two dogs in the group over the first 12 hours. However, from the 18 hour datapoint until the completion of the data collection for this dog, plasma and RBC concentrations were significantly elevated, more than three times the concentrations of the other two dogs in the group.

The highest mean concentrations from Group A were detected in the liver (1.34 ppm) and mesenteric fat (0.945 ppm). As was observed in the plasma and RBCs, the concentrations of radioactivity in the liver and fat from Animal No. H09704 were significantly higher than, the concentrations of similar samples from the other two dogs (again, approximately three times higher). The reason for the higher concentrations of radioactivity in plasma, RBCs, and tissues was not determined.

The recovery of radioactivity in the excreta from the animal in question was midrange to the other dogs (84.4% of dose administered versus 65.8% and 86.0% for the other two dogs, respectively). Because the dog was in the top cage, contamination from another cage was not possible. There were no observations made of unusual events for this animal. It is possible that following the 12 hour blood collection the dog ingested some of its own feces, resulting in the increase in plasma, RBC and tissue concentrations (essentially a redose). It is also possible that the dog vomited and aspirated the vomitus into the lungs. The exposure of the test material in the stomach contents to the thin-membrane, blood-rich lung tissue could have resulted in the increased uptake of the test material into the bloodstream. The ingestion of feces or aspiration of vomit could explain why there was no effect on the overall recovery of radioactivity but that higher recoveries of radioactivity in the urine/feces collections were shifted to later times.

There were no radioactive residues detected in any Group C (control) samples.

The Group В dogs were dosed with test material at 15 mg/kg and were sacrificed at the Cmax from the Group A plasma levels (2 hours after dosing). Highest mean residue concentrations were seen in the liver (4.45 ppm), mesenteric fat (2.80 ppm), plasma (0.999 ppm), and RBCs (0.413 ppm). The residues in the aqueous humor, eye, and eye remainder were 0.061 ppm, 0.106 ppm, and 0.131 ppm, respectively. Urine and feces were not collected.

Reference 4

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

Objective of study:

excretion

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance name: [14C-phenoxyphenyl]DPX-JE874 (POP label)
Lot # 3048-237
Purity: Not reported

Substance name: [14C-phenylamino]DPX-JE874 (PA label)
Lot# 3048-238
Purity: Not reported

Radiolabelling:

yes

Species:

rat

Strain:

other: Crl:CD/BR (Sprague-Dawley) albino

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: 1% sodium carboxymethylcellulose

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

5 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

5

Control animals:

no

Type:

excretion

Results:

A large portion of administered dose (30-39% AD) was rapidly excreted in the bile, occurring between 1 to 10 h post-dose. The average urinary excretion of radiolabel ranged from 2-6% AD. Most of the administered radiolabel, 56-65% was excreted in feces.

Details on absorption:

The amount of radiolabel that was absorbed was calculated as the sum of the radiolabel in bile, urine, cage wash, carcass and blood. A significant portion of the administered radiolabel was absorbed. The amount of radiolabel absorbed was 37-38% AD in the [14C-PA]test substance treated males and females, and 37-41% AD in the [14C-POP]test substance treated males and females. There were no Statistical differences between sexes (for a given label) or between labels (for a given sex) with respect to the extent of absorption.

Details on distribution in tissues:

Radiolabel in the Blood and Carcass: The amount of radioactivity that was found in the blood at the termination of the experiment (48 h) was statistically different in the PA and POP label groups. The amount of [14C] residues in the blood at termination was 0.22 and 0.31% AD in the PA males and females, respectively, and 0.03% AD in both the POP males and females. The average amount of radiolabel found in the carcasses at termination was low, ranging from 0.4-3 % AD.

Details on excretion:

Bile: A large portion of the administered dose (30-39% AD) was rapidly excreted in the bile, occurring between 1 to 10 h post-dose. There were no statistical differences between sexes (for a given label) or between labels (for a given sex) with respect to the rate/extent of radiolabel excretion in the bile, with one exception. An apparent difference in the amounts of radiolabel excreted in the bile of the PA and POP males was observed (31 vs. 39%).

Urine: The average urinary excretion of radiolabel ranged from 2-6% AD. There were no statistical differences between sexes (for a given label) or between labels (for a given sex) with respect to the elimination of radioactivity via urine.

Feces: Most of the administered radiolabel, 56-65%, was excreted in the feces. There were no statistical differences between sexes (for a given label) or between labels (for a given sex) with respect to rate and extent of fecal excretion.

Metabolites identified:

yes

Remarks:

Greater than 80%, (ranged from 82-98%) of the radioactive components in the bile was characterized. Conjugates of IN-KZ007, and catechol and conjugates of IN-KZ007 and IN-ML436 were the major metabolites in bile of the [14C-PA] and [14C-POP], respectively

Details on metabolites:

Feces: The only radioactive component present in the fecal extracts coeluted with the synthetic standard of test substance.

Bile: Selected bile samples from the PA and POP labels were analyzed using HPLC. None of the peaks observed matched the available metabolite standards. Bile samples were then treated with ß-glucuronidase/sulfatase to release glucuronide and sulfate conjugates to the corresponding non-conjugated metabolites, which were analyzed by HPLC/RAD using HPLC. The chemical nature of the enzyme products was characterized by HPLC co-chromatography with authentic standards. The treated bile samples were coinjected with standards to demonstrate coelution of the products of the enzyme reactions with standards. For the POP label, the following standards were included: IN-ML436, IN-MN967, IN-KZ000, IN-ML815, IN-KZ534, IN-KZ007, IN-KZ532, and DPX-JE874. For the PA label, the following standards were included: catechol, IN-ML436, IN-ML815, IN-KZ534, and IN-KZ007. For the PA label, catechol, IN-ML815, IN-KZ534, IN-KZ007, and IN-KZ532 were demonstrated. For the POP label, IN-ML436, INML967, IN-KZ000, IN-ML815, IN-KZ534, IN-KZ007, and IN-KZ532 were demonstrated.

Conclusions:

Metabolism of the test substance occurred via the hydroxylation of the phenoxyphenyl and phenylamino rings, hydrolysis of the oxazolidinedione moiety, cleavage of the phenylamino ring, and by combinations of these pathways. Further conjugation of these primary metabolites also occurred.

Executive summary:

The absorption, excretion and metabolism of the test substance in biliary-cannulated male and female Sprague-Dawley rats was examined. The study was conducted following US EPA guideline 85-1. These metabolic parameters were examined using [14C-phenoxyphenyl] (POP) and [14C-plienylamino] (PA) test substance following a single oral dose of 5 mg/kg. Five rats were included for each sex and label group. The animals were terminated 48 h post-dose.

A large portion of the administered dose (AD) was rapidly excreted in the bile, with the peak hourly excretion occurring between 1 to 10 h post-dose. The average amount of radiolabeł excreted in the bile ranged from 30-39 % AD. Only a small amount of radiolabel, averaging from 2-6% AD, was excreted in the urine. Most of the administered radiolabel, averaging from 56-65% AD, was excreted in the feces. At the 48-h termination, an average of only 0.4-3% AD remained in the carcasses. The amount of radiolabel absorbed (defined as the sum of radiolabel in the bile, urine, cage wash, blood and carcass) was 38% AD and 37% AD in the [14C-PA]test substance treated males and females, respectively, and 41 and 37% AD in the [14C-POP]test substance treated males and females, respectively. In generał, there were no statistically significant differences (p <0.05) between sexes (for a given label) or between labels (for a given sex) with respect to any of the above parameters.

Fecal and bile extracts from both the phenoxyphenyl and phenylamino labels were examined. Urine was not analyzed due to the low levels of the administered dose present.

Unmetabolized test substance was the only radiolabeled component detected in the fecal extract, amounting to 26-58% of the administered dose.

The test substance was not detected in the bile samples. Several metabolic products were detected. These are polar compounds, possibly conjugated products, which did not co-chromatograph with standards of known test substance metabolites. Bile samples were treated with ß-glucuronidase/sulfatase and the released metabolites were then identified by HPLC co-chromatography and confirmed by mass spectrometry with or without derivatization. Qualitatively, conjugates of IN-KZ007, IN-KZ532, IN-KZ534, IN-ML815, and catechol were observed in the bile of the [ I4C-P A]test substance-treated rats. Conjugates of IN-KZ007, IN-KZ532, IN-KZ000, IN-KZ534, IN-MN967, IN-ML815, and IN-ML436 were observed in the bile of the [I4C-POP]test substance-treated rats.

Quantitatively, conjugates of IN-KZ007 and catechol, and conjugates of IN-KZ007 and IN-ML436 were the major metabolites in bile of the [I4C-PA] and [14C-POP]test substance-treated rats, respectively.

Metabolism of test substance occurred via the hydroxylation of the phenoxyphenyl and phenylamino rings, hydrolysis of the oxazolidinedione moiety, cleavage of the phenylamino ring, and by combinations of these pathways. Further conjugation of these primary metabolites also occurred.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Radioactive test substance
[14C-POP]DPX-JE874
Lot #: 2675-268
Radiochemical purity: >98%
Specific activity: 22.3 mCi/mmole

[14C-PA]DPX-JE874
Lot #: 2868-107
Radiochemical purity: >98%
Specific activity: 20.5 mCi/mmole

Non-Radioactive test substance
DPX-JE874
Lot # DPX-JE874-133 (Drum 10)
Purity: >96%

Radiolabelling:

yes

Species:

rat

Strain:

other: Crl:CD/BR ( Sprague-Dawley) albino

Details on species / strain selection:

Crl:CD/BR rats were used for this study because they have been used in the subchronic and chronic toxicology studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan
- Age at study initiation: Males: 7-8 weeks; Females: 9-10 weeks; Multiple dose study: 6-8 weeks
- Weight at study initiation: The average body weight 201 ± 18 g
- Housing: Prior to dosing, rats were housed individually in stainless steel cages for a minimum period of 7 days. After dosing with [14C]test substance, animals in the pilot group were housed individually in glass metabolism chambers designed for the separate collection of urine, feces and expired air. Polycarbonate metabolism chambers and stainless steel cages with raised wire mesh floors were used when the monitoring of 14CO2 in the expired air was not required (all groups except the pilot group). The animals receiving multiple daily oral doses were housed individually in stainless steel cages during the first week of the nonradiolabeled test substance dosing. These animals were then transferred to polycarbonate metabolism chambers for the collection of excreta during the remaining nonradiolabeled test substance dosing regimen. Each animal was transferred to a second (clean) polycarbonate metabolism chamber immediately after radiolabeled test substance dosing.
- Diet: ad libitum (except for an overnight predose fast prior to administration of radiolabeled material)
- Water: Tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 2°C
- Humidity: 40-70%
- Air changes: 10-15 per hr
- Photoperiod: 12 hrs dark /12 hrs light

Route of administration:

oral: unspecified

Vehicle:

other: 1% sodium carboxymethyl cellulose/0.01 M ammonium acetate

Details on exposure:

Oral doses (5 and 100 mg/kg) were prepared as a suspension in 1% sodium carboxymethyl cellulose in 0.01 M ammonium acetate/HPLC grade water.

Duration and frequency of treatment / exposure:

Each rat received a single oral dose of either [14C-PA]test substance or [14C-POP]test substance.
In the case of the multiple dose group (Group G), the radiolabeled dose was preceded by 14 consecutive daily doses of non-radiolabeled test substance.

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group P (Pilot)
[14C-PA]test substance

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group P (Pilot)
[14C-POP]test substance

Dose / conc.:

5 mg/kg bw/day

Remarks:

Group A (Kinetics)
[14C-PA]test substance

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group B (Kinetics)
[14C-POP]test substance

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group C (Kinetics)
[14C-PA]test substance

Dose / conc.:

5 mg/kg bw/day

Remarks:

Group D (Metabolism)
[14C-PA]test substance

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group E (Metabolism)
[14C-POP]test substance

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group F (Metabolism)
[14C-PA]test substance

Dose / conc.:

5 mg/kg bw/day

Remarks:

Group G (Metabolism)
[14C-PA]test substance

Dose / conc.:

5 mg/kg bw/day

Remarks:

Group H (Distribution)
[14C-PA]test substance

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group I (Distribution)
[14C-PA]test substance

No. of animals per sex per dose / concentration:

Group P*: 2/sex
Group A, B, C, H*, I*: 4/sex
Group: D, E, F, G: 5/sex
Group J (control): Minimum 2/sex
(*Includes 2 groups)

Control animals:

yes, concurrent vehicle

Details on study design:

Two different test substances were examined. [14C]test substance was labeled with Carbon-14 at the phenoxyphenyl and phenylamino ring moieties. [14C]test substance was administered at the following target dose levels: Oral High Dose: 100 mg/kg & Oral Low Dose: 5 mg/kg.
The high dose level was chosen on the basis that it was a level at which slight toxicological effects could be expected. The low dose level corresponds to an approximate no-effect level.
This study involved single oral (low and high dose level) and multiple oral (low dose level) dose administrations. The target radioactive dose to each animal from the main treatment groups was approximately 30-50 µCi.

Details on dosing and sampling:

- Tissues and body fluids sampled: Urine, faeces, expired air, blood, plasma and tissues (kidneys, liver, fat, gonads, uterus, muscle, heart, lungs, spleen, brain, bone, bone marrow, adrenals, thyroid, skin, GI tract and the remaining carcass)

Statistics:

Statistical comparisons were performed using Student's t-test.

Preliminary studies:

Results of Pilot study:
The 14C-radiolabel is stable at both the phenoxyphenyl and phenylamino portions of the parent molecule, and therefore, expired air of the treated animals need not be collected/monitored in the main treatment groups.
A termination time of 120 hours would be sufficient to ensure that ≥ 90 % of the administered radioactive dose is excreted.
Cleavage of the test substance is likely between the phenoxyphenyl and phenylamino moieties, with greater amounts of tissue residues being associated with the phenylamino portion of the molecule. The focus of this rat metabolism and pharmacokinetic study will be on the phenylamino portion of the molecule.
Most of the administered dose is excreted as unchanged test substance in the feces.

Type:

absorption

Results:

Absorption half-life of [14C-PA]test substance increased from 0.8-1.2 hours to 3.5-7.1 hours as the dose level increased from 5 to 100 mg/kg. The absorption half-lives of [14C-POP]test substance at the 100 mg/kg appeared to be be rapid, at 0.4 to 1.4 hrs.

Type:

distribution

Results:

At the 120-hour sampling interval, no specific retention of 14C-residues was observed in any of the examined tissues. Accumulation in the various organs and tissues were not observed, with the tissue/blood ratio <1.

Type:

excretion

Results:

Greater than 90% of the administered radioactivity was recovered in the fecal excreta within 120 hours postdosing. Urinary elimination generally accounted for less than 10% of the administered dose.

Type:

metabolism

Results:

Unmetabolized [14C]test substance was the major component recovered from the fecal excreta. Mono- (IN-KZ007) and dihydroxylated (IN-KZ534) test substance were identified as primary metabolites, recovered only from the feces.

Details on absorption:

The initial absorption of [14C-PA]test substance following oral dosing of 5 mg/kg was rapid. The apparent absorption half-lives of total [14C]-residues in the whole blood and plasma were approximately 0.8 to 1.2 hours.
The absorption half-life of [14C-PA]test substance increased from 0.8-1.2 hours to 3.5-7.1 hours as the dose level increased from 5 to 100 mg/kg. However, the absorption half-lives of [14C-POP]test substance at the 100 mg/kg high dose level appeared to be rapid, at approximately 0.4 to 1.4 hours. This observation suggested the potential absorption of the metabolite(s) derived from the phenylamino moiety.

Details on distribution in tissues:

Liver and body fat appeared to be the two primary tissues for the distribution of [14C-PA]test substance equivalent residues during the initial phase after oral exposure (at tcmax).
Tissue residues were eliminated rapidly. At the t cmax/2sampling interval, liver was the only tissue containing significant [14C-PA]test substance equivalent residues, where tissue/blood ratio was >1.0. At the 120-hour sampling interval, no specific retention of 14C-residues was observed in any of the examined tissues.

Details on excretion:

Greater than 90% of the administered radioactivity was recovered in the fecal excreta within 120 hours postdosing. Urinary elimination generally accounted for less than 10% of the administered dose. Elimination was rapid; the majority of the administered dose was eliminated usually within the initial 24 hours. 14CO2 was not detected in the expired air.

Test no.:

#1

Toxicokinetic parameters:

other: Absorption half life

Remarks:

[14C-PA]test substance: ~0.8-1.2 hours (5 mg/kg); ~3.5-7.1 hours (100 mg/kg) [14C-POP]test substance: ~0.4-1.4 hours (100 mg/kg);

Metabolites identified:

yes

Details on metabolites:

Unmetabolized [14C]test substance was the major component recovered from the fecal excreta. Mono- (IN-KZ007) and dihydroxylated (IN-KZ534) test substance were identified as primary metabolites, recovered only from the feces. Cleavage products, including IN-KZ000 sulfate and 4-acetoxyanxline were the primary urinary metabolites recovered from the [14C-POP] and [14C-PA] test substance treated animals, respectively.

Conclusions:

Greater than 90% of the administered radioactivity was recovered in the fecal excreta within 120 hours postdosing. Urinary elimination generally accounted for less than 10% of the administered dose. Elimination was rapid; the majority of the administered dose was eliminated usually within the initial 24 hours. 14CO2 was not detected in the expired air.
Unmetabolized [14C]test substance was the major component recovered from the fecal excreta. Mono- (IN-KZ007) and dihydroxylated (IN-KZ534) test substance were identified as primary metabolites, recovered only from the feces.
Cleavage products, including IN-KZ000 sulfate and 4-acetoxyanxline were the primary urinary metabolites recovered from the [14C-POP] and [14C-PA] test substance treated animals, respectively.
Accumulation of [14C-PA] or [14C-POP] test substance equivalent residues in the various organs and tissues were not observed, with the tissue/blood ratio <1.
No significant differences in the overall metabolic fate of the test substance in the male vs the female test animals, high vs low dose, single vs multiple dosing regimen were observed.

Executive summary:

The absorption, distribution, metabolism and elimination of the test substance was examined according to the guideline EPA 85-1. These metabolic parameters were examined using [14C-Phenoxyphenyl (POP)] and [14C-Phenylamino (PA)] test substance in male and female Sprague-Dawley rats following a single or multiple oral dosing at the 5 or 100 mg/kg dose level. Greater than 90% of the administered radioactivity was recovered in the fecal excreta within 120 hours postdosing. Urinary elimination generally accounted for less than 10% of the administered dose. Elimination was rapid; the majority of the administered dose was eliminated usually within the initial 24 hours. 14CO2 was not detected in the expired air.

Unmetabolized [14C]test substance was the major component recovered from the fecal excreta. Mono- (IN-KZ007) and dihydroxylated (IN-KZ534) test substance were identified as primary metabolites, recovered only from the feces. Cleavage products, including IN-KZ000 sulfate and 4-acetoxyanxline were the primary urinary metabolites recovered from the [14C-POP] and [14C-PA] test substance treated animals, respectively.

Accumulation of [14C-PA] or [14C-POP] test substance equivalent residues in the various organs and tissues were not observed, with the tissue/blood ratio <1. Whole blood and plasma pharmacokinetic parameters were calculated to provide a description of the behavior and metabolic fate of the test substance in the test animals. Blood residue data showed the potential of binding of [14C-PA]test substance equivalent residues to the red blood cells.

No significant differences in the overall metabolic fate of the test substance in the male vs the female test animals, high vs low dose, single vs multiple dosing regimen were observed.

Description of key information

Rats

The absorption, distribution, metabolism, and elimination of ¹⁴C-famoxadone [¹⁴C-phenoxyphenyl (POP) and ¹⁴C‑phenylamino (PA)] was examined in male and female rats following single or multiple oral dosing at 5 and 100 mg/kg bw.  ¹⁴CO₂ was not observed in the expired air collected from pilot groups.  Between 88.8 and 96% of the administered radioactive dose was recovered in the faecal excreta, with over 80% within the first 24 hours after dosing.  Urinary elimination ranged from 3% in the 100 mg/kg bw dose groups to 12% in the 5 mg/kg bw dose groups.  Over 70% of the administered dose that was eliminated in urine (<10%) occurred in the initial 24 hours.  No significant difference was found in the elimination profile under single and multiple dose regimens.

Unmetabolized ¹⁴C-famoxadone was the major component recovered in the faeces, with the hydroxylated metabolites (IN-KZ007 or IN-KZ534) identified as major metabolites (up to 13% of the administered dose).  In urine only hydrolytic (IN-JL856) and hydrolysis cleavage products (IN‑KZ000, IN‑BY759, and IN-H3310) were detected (up to 7% of the administered dose).  After 36‑48 hours, tissue/blood concentration ratio were ≤0.5 for all tissues, except liver which showed ratios of 1.8 and 1 in the lower and higher dose groups, respectively.

The biliary excretion of famoxadone was investigated in bile duct-cannulated male and female rats following a single oral dose of 5 mg/kg bw ¹⁴C-POP or ¹⁴C-PA labelled test material.  Urine, bile, and faeces were collected continuously for 48 hours post-dosing.  The average quantity of the administered dose excreted in the bile ranged from 30 to 39% and in the faeces from 56 to 65%.  The average absorbed dose was 40% (sum of radioactivity measured in the bile, urine, cage wash, blood, and carcass).  There was no difference between males or females for absorption and elimination in the bile or excreta.

Famoxadone was the only radio-labelled component in solvent extracts of faeces, and it was not detect in bile samples.  The main metabolites released in bile samples treated with ß‑glucuronidase/sulfatase were: IN-KZ007 (up to 5.1% of the administered dose; catechol (1,2 dihydroxybenzene or IN-03490, up to 4.6%), and IN-ML436 (up to 3.6%).  IN-MN967 and IN‑ML815 were minor metabolites released from the bile by enzyme treatment accounting for up to 0.05% and 1.8% of the dose, respectively.

Other Species:

The absorption, distribution, and elimination of famoxadone were studied in male beagle dogs dosed orally with 15 mg/kg bw ¹⁴C-phenylamino labelled test material.  Most of the radioactivity was excreted within 96 hours after dosing.  Urine, faeces, cage washes and cage wipes accounted for 78.8% of the administered dose (range 65.8% to 86.0%).  The T_max for radioactive residues in plasma and RBCs was 2 and 4 hours, respectively.  The depletion of radioactive residues from the plasma and RBCs was slower than for urine and faeces.  The t½ ranged from 67 to 75 hours for plasma and 146 to 159 hours for RBCs.  The concentration of radioactive residues in tissues was highest in fat and liver.  There were no measurable residues found in the whole eye, the aqueous humour of the eye, and the remainder of the eye (following removal of the aqueous humour).

Analysis of RBCs, plasma, liver, and fat taken 2 hours after a single oral dose of ¹⁴C-phenylamino famoxadone in the ADME study in beagle dogs was under taken.  The circulating metabolites in plasma which were quantified included parent famoxadone and the metabolites IN-KZ007, IN-JL856, and IN-ML815.  The major component in plasma was IN-KZ007.  These three metabolites, in addition to parent, were also present in RBCs.  Some recycling of these metabolites was evident, leading to multimodal concentration/time profiles.  Other metabolites were present in plasma but their identity could not be elucidated.  Extracts of liver and fat were shown to contain predominantly famoxadone with lesser amounts of IN-KZ007.  No further metabolites were noted in fat, but liver contained several other unidentified metabolites.  A complex pattern of radiolabelled metabolites was observed in urine and faeces.  Urine contained mainly polar components, none of which could be identified with reference to known metabolites of the phenylamino moiety of famoxadone; there was no evidence to suggest that glucuronide or sulphate conjugates were present.  Faeces extracts contained mainly famoxadone at early collection times, but more metabolites were formed at later times with included IN-ML815, IN-KZ534, IN‑KZ532, IN-KZ007, and IN-JL856.

Additional information

A study was conducted to investigate the in vivo dermal penetration of [14C]JE-874 (and/or its metabolites) in female laboratory rats, following a single dermal administration of [14C]DPX-MC444-11 formulation at two dose levels: as the undiluted emulsifiable concentrate and as an aqueous dilution of the concentrate equivalent to approximately ten times that of the field application solution. Overall, limited dermal penetration of test substance was observed. The majority of administered radioactivity was removed at 8 h or 24 h by washing of the application site. Maximal dermal absorption occurred within 24 h after the initial application. Dermal absorption (expressed as the percent of the applied radioactivity) for the aqueous solution was approximately twice that for the formulation concentrate. Negligible amounts (<1%) of the applied radioactivity was recovered in the urine. Faeces accounted for approximately 6% of the applied radioactivity. The majority of the applied radioactivity was removed in the skin wash. Analysis of the skin washes, by HPLC, following 24 h exposure showed the presence of a single radioactive component with a retention time consistent with that of the test substance. Excreted radioactivity was largely eliminated as test substance in the faeces with some IN-KZ007 also present. In conclusion, following a single dermal administration of [14C]test substance, limited dermal penetration was observed, with the majority of excreted radioactivity eliminated in the faeces. Washing the application site effectively removed the majority of applied radioactivity.