picoxystrobin (ISO); methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

99.8% purity for the unlabeled test substance
>97% purity for the radiolabeled pyridyl and phenyl moieties of the test substance

Radiolabelling:

yes

Species:

rat

Strain:

other: Alpk:APf SD

Details on species / strain selection:

The Alpk:Apr SD strain of rat was selected because this strain was used during other toxicology studies with this test substance and since substantial background information is available for its use in the testing laboratory. The oral route of administration was used to represent a possible route of human exposure.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Not reported
- Weight at study initiation: 180-208 g
- Housing: The rats were housed in groups of the same sex in stock rat cages. Following acclimatization, they were transferred to glass metabolism cages, in which they were individually housed and acclamatised for approximately 1 day prior to dosing.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): At least 15 changes/hour, giving positive pressure with respect to the access corridor
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 600

Duration and frequency of treatment / exposure:

24 hours

Dose / conc.:

10 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

2 male and 2 females

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

No

Details on study design:

- Dose selection rationale: The dose level was selected to represent a 'no-effect' dose following single oral administration.

Details on dosing and sampling:

The [14C]-pyridyl dosing solution was prepared by dissolving 8.6 mg of unlabeled test substance and 9.6 MBq (equivalent to 1.9 mg) of the [14C]-pyridyl moiety in the dosing vehicle to give a final weight of 11.2 g and concentrations of 0.9 mg/g and 0.9 MBq/g of dosing solution. The specific activity of the test substance in the dosing solution was 912.8 kBq/mg of the test substance.

The [14C]-phenyl dosing solution was prepare by dissolving 8.6 mg of unlabeled test substance and 9.8 MBq (equivalent to 1.8 mg) of the [14C]-phenyl moiety in the dosing vehicle to give a final weight of 11.3 g and concentrations of 0.9 mg/g and 0.9 MBq/g of dosing solution. The specific activity of the test substance in the dosing solution was 945.5 kBq/mg of the test substance.

After dosing, the animals were returned to separate metabolism cages. Urine only was collected at 6 hours after dosing and urine and feces were separately collected at 12 and 24 hours from all animals. The exhaled air from each animal was passed through two sequential Nilox© columns (A and B) each containing 2 M sodium hydroxide solution followed by a single trap containing 2 M hydrochloric acid and a water trap and finally through a single Draegar tube pre-packed with activated charcoal. Subsamples of the contents of each trap were removed for radiochemical analysis at 12 and 24 hours after dosing.

Statistics:

The use of one rat of each sex precludes any statistical analysis.

Details on distribution in tissues:

For the [14C]-pyridyl moiety, in the WBA sections, the majority of the radioactivity was observed in the intestinal tract contents of both sexes. Tissue radiolabeling was apparent in the liver with lower levels in the kidneys. Lower intensities of radioactivity were observed in all other tissues.

For the [14C]-phenyl moiety, in the WBA sections, the highest intensity of radioactivity was observed in the gastrointestinal tract contents of both sexes and also the stomach mucosa of the male rat. Tissue radiolabelling was apparent in the liver and kidneys, with lower intensities of radioactivity observed in all other tissues.

Details on excretion:

Twenty-four hours after dosing with the [14C]-pyridyl moiety, the male rat excreted 21.0% of the administered dose in the urine and 13.4% in the faeces. The similarly dosed female rat excreted 25.0% of the dose in the urine and 19.1% in the faeces over the same period. For both sexes, less than 0.4% of the administered dose was exhaled as volatile metabolites over 24 hours after dosing.

Twenty-four hours after dosing with [14C]-phenyl moiety, the male rats excreted 17.8% of the administered dose in the urine and 19.6% in the faeces. The female rats excreted 30.2% of the administered dose in the urine and 19.6% in the faeces over the same period. For both sexes, less than 0.1% of the dose was exhaled as volatile metabolites.

Conclusions:

Based upon the small number of animals used and the short duration of this study, no conclusions can be made about the rate or extent of absorption. However, for both the [14C]-pyridyl and [14C]-phenyl moieties, negligible proportions of the dose were metabolized to volatile exhaled metabolites. Twenty-four hours after dosing the greatest intensity of radiolabeling was in the contents of the gastrointestinal tract with radiolabeling apparent in the liver with lesser amounts in the kidneys and lower levels in other tissues.

Executive summary:

Male and female rats were administered a single oral dose of 10 mg/kg of the 14C radiolabeled pyridyl or phenyl moiety of the test substance. The excretion of radioactivity was monitored in urine, feces and exhaled air. Twenty-four hours after dosing, the rats were killed to investigate the distribution of radioactivity using whole body autoradiography (WBA).

Twenty-four hours after dosing with the [14C]-pyridyl moiety, the male rat excreted 21.0% of the administered dose in the urine and 13.4% in the feces. The similarly dosed female rat excreted 25.0% of the dose in the urine and 19.1% in the feces over the same period. For both sexes, less than 0.4% of the administered dose was exhaled as volatile metabolites over 24 hours after dosing. In the WBA sections, the majority of the radioactivity was observed in the intestinal tract contents of both sexes. Tissue radiolabeling was apparent in the liver with lower levels in the kidneys. Lower intensities of radioactivity were observed in all other tissues.

Twenty-four hours after dosing with [14C]-phenyl moiety, the male rats excreted 17.8% of the administered dose in the urine and 19.6% in the feces. The female rats excreted 30.2% of the administered dose in the urine and 19.6% in the feces over the same period. For both sexes, less than 0.1% of the dose was exhaled as volatile metabolites. In the WBA sections, the highest intensity of radioactivity was observed in the gastrointestinal tract contents of both sexes and also the stomach mucosa of the male rat. Tissue radiolabelling was apparent in the liver and kidneys, with lower intensities of radioactivity observed in all other tissues.

Reference 2

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:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

99.8% purity

Radiolabelling:

yes

Species:

rat

Strain:

other: Alpk:Apf SD

Details on species / strain selection:

The rat was selected as a species recommended in the regulatory guidelines. The Alpk:APf SD strain of rat was selected because this strain was used during other toxicology studies with this test substance and since substantial background information is available for its use in the testing laboratory. The oral route of administration was used to represent a possible route of human exposure.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Not reported
- Weight at study initiation: 180-208 g
- Housing: The rats were housed in groups of the same sex in stock rat cages. Following acclimatization, they were transferred to stainless steel metabolism cages, in which they were individually housed and acclimatized for approximately one day prior to dosing.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): At least 15 changes/hour, giving positive pressure with respect to the access corridor
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 600

Details on exposure:

Five male and five female rats were each administered a single oral dose of 100 mg/kg of the [14C]-labeled test substance.

Duration and frequency of treatment / exposure:

Single oral dose. The excretion of radioactivity in urine and faeces was monitored for 120 hours after dosing.

Dose / conc.:

100 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

5 males and 5 females

Control animals:

no

Positive control reference chemical:

No

Details on study design:

- Dose selection rationale: The dose selected to represent a high dose level. It exceeds the 'no-effect' dose level selected for the corresponding excretion and tissue distribution study by ten fold.

Details on dosing and sampling:

The dose was prepared by dissolving 496 mg of the unlabeled test substance and 26.9 MBq (equivalent to 4.84 mg) of the [14C]-labeled test substance in the dose vehicle to give a final volume of 10 mL and concentrations of 44.3 mg/g of test substance and 2.39MBq/g of dose preparation. The specific activity of the test substance in the dose preparation was 53.8 MBq/g of the test substance. Hence when administered at a dose rate of nominally 2 mL/kg, this represented a nominal dose level of 100 mg/kg of the [14C]-labeled test substance and 5.38 MBq/kg.

Triplicate weighed aliquots of the dose preparation were diluted volumetrically in acetone and triplicate aliquots of each dilution were taken for liquid scintillation counting.

The radiochemical purity of the [14C]-labeled test substance was determined, prior to and following formulation in the dose vehicle, by thin layer chromatography (TLC) using silica-gel plates (60F254) in solvent systems 1-4:

1) Diethyl ether:dichloromethane (1:4 v/v)
2) Hexane:diethyl ether (1:9 v/v)
3) Hexane:ethyl acetate (1:1 v/v)
4) Chloroform:methanol:formic acid:water (75:25:4:4v/v/v/v)

Dilutions of the [14C]-labeled test substance were co-chromatographed with the unlabeled test substance. Radioactivity on the TLC plates was measured using a Berthold Tracemaster linear analyser. The unlabeled test substance was visualized under UV light at absorption 254 nm. Typical Rf values for the test substance were 0.79, 0.74, 0.55 and 1.0 for systems 1,2, 3 and 4 respectively.

The purity of the [14C]-labeled test substance was determined, prior to and following formulation in the dose vehicle, by HPLC using the following conditions: An Alltima C18, 5 μm, reverse phase column (150 x 4.6 mm, Alltech Associates Applied Science Ltd, Camforth, Lancashire, UK) was eluted with water:acetonitrile (4:6 v/v with 0.1% acetic acid in each) at a flow rate of 1 mL/minute. Radioactivity was detected using an on-line flow detector and with UV absorption at 254 nm. Dilutions of the radiolabeled test substance were co-chromatographed with the unlabeled test substance. A typical retention time for the test substance was 10.8 minutes.

Statistics:

No

Details on distribution in tissues:

All tissue concentrations were low at the termination of this study. With the exception of liver, kidneys, GI tract, blood and plasma, group mean tissue concentrations were less than 0.65 μg equiv/g for males and 0.85 μg equiv/g for females. All tissue concentrations of radioactivity with the exception of liver, were lower in males than females. The amount of administered radioactivity present in the tissues and residual carcass of males and females was 0.78% and 0.91% respectively.

Details on excretion:

Over 120 hours, males and females both excreted mean totals of 92% of administered radioactivity. Urinary excretion accounted for mean totals of 18% and 26% for males and females respectively with females showing a higher rate of excretion in urine than males throughout the study. The excretion of radioactivity was predominantly in faeces accounting for mean totals of 74% of the dose in males and 65% in females.

Metabolites identified:

no

Conclusions:

Following a single oral administration of nominally 100 mg/kg of the [14C]-labeled test substance to male and female rats, excretion was extensive and predominantly in faeces. Males excreted a greater proportion of the radioactivity in faeces and a lower proportion in urine than females. Tissue concentrations were low for both sexes.

Executive summary:

Five male and five female rats were each administered a single oral dose of 100 mg/kg of the [14C]-labeled test substance. The excretion of radioactivity in urine and faeces was monitored for 120 hours after dosing. After this period, the rats were killed and the remaining radioactivity was measured in blood, selected tissues and in the residual carcasses.

 

Over 120 hours, males and females both excreted mean totals of 92% of administered radioactivity. Urinary excretion accounted for mean totals of 18% and 26% for males and females respectively with females showing a higher rate of excretion in urine than males throughout the study. The excretion of radioactivity was predominantly in faeces accounting for mean totals of 74% of the dose in males and 65% in females. At termination of the study, the mean percentage of dose present in the gastrointestinal tract contents was 0.8% or less for both sexes.

 

All tissue concentrations were low at the termination of this study. With the exception of liver, kidneys, GI tract, blood and plasma, group mean tissue concentrations were less than 0.65 μg equiv/g for males and 0.85 μg equiv/g for females. All tissue concentrations of radioactivity with the exception of liver, were lower in males than females. The amount of administered radioactivity present in the tissues and residual carcass of males and females was 0.78% and 0.91% respectively.

Reference 3

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:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

99.8% purity

Radiolabelling:

yes

Species:

rat

Strain:

other: Alpk:Apf SD

Details on species / strain selection:

The rat was selected as a species recommended in the regulatory guidelines. The Alpk:APf SD strain of rat was selected because this strain was used during other toxicology studies with this test substance and since substantial background information is available for its use in the testing laboratory. The oral route of administration was used to represent a possible route of human exposure.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Not reported
- Weight at study initiation: 180-208 g
- Housing: The rats were housed in groups of the same sex in stock rat cages. Following acclimatization, they were transferred to stainless steel metabolism cages, in which they were individually housed and acclimatized for approximately one day prior to dosing.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): At least 15 changes/hour, giving positive pressure with respect to the access corridor
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 600

Duration and frequency of treatment / exposure:

One single oral gavage dose with a 120-hour period for collection of urine and faeces.

Dose / conc.:

10 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

4 males and 4 females for Sample A followed by 1 male and 1 female for Sample B

Control animals:

no

Positive control reference chemical:

No

Details on study design:

- Dose selection rationale: The dose level was selected to represent a 'no-effect' dose following a single oral administration.

Details on dosing and sampling:

Dose A was prepared by dissolving 22.7 mg of unlabeled test substance and 13 MBq (equivalent to 2.33 mg) of the [14C]-radiolabeled test substance in the dose vehicle to give a final volume of 5 mL and concentrations of 4.41 mg/g of the test substance and 2.28 MBq/g of dose preparation. The specific activity of the test substance in the dose preparation was 518 MBq/g of the test substance. Hence when administered at a dose rate of nominally 2 mL/kg, this represented a nominal dose level of 10 mg/kg of the [14C]-radiolabeled test substance and 5.2 MBq/kg.

Dose B was prepared by dissolving 68.7 mg of unlabeled test substance and 33.4 MBq (equivalent to 6 mg) of the [14C]-radiolabeled test substance in the dose vehicle to give a final volume of 15 mL and concentrations of 4.45 mg/g of the test substance and 1.99 MBq/g of dose preparation. The specific activity of the test substance in the dose preparation was 447 MBq/g of the test substance. Hence when administered at a dose rate of nominally 2 mL/kg, this represented a nominal dose level of 10 mg/kg of the [14C]-radiolabeled test substance and 4.5 MBq/kg.

The radiochemical purity of the [14C]-labeled test substances A and B was determined, prior to and following formulation in the dose vehicle, by thin layer chromatography (TLC) using silica-gel plates (60F254) in solvent systems 1-4:

1) Diethyl ether:dichloromethane (1:4 v/v)
2) Hexane:diethyl ether (1:9 v/v)
3) Hexane:ethyl acetate (1:1 v/v)
4) Chloroform:methanol:formic acid:water (75:25:4:4v/v/v/v)

Dilutions of the [14C]-labeled test substance were co-chromatographed with the unlabeled test substance. Radioactivity on the TLC plates was measured using a Berthold Tracemaster linear analyser. The unlabeled test substance was visualized under UV light at absorption 254 nm. Typical Rf values for the test substance A were 0.73, 0.70, 0.58 and 0.95 for systems 1,2, 3 and 4 respectively and for test substance B, 0.79, 0.74, 0.55, and 1.00, respectively.

The purity of the [14C]-labeled test substance was determined, prior to and following formulation in the dose vehicle, by HPLC using the following conditions: An Alltima C18, 5 μm, reverse phase column (150 x 4.6 mm, Alltech Associates Applied Science Ltd, Camforth, Lancashire, UK) was eluted with water:acetonitrile (Sample A, 3:7 v/v and Sample B 4:6 v/v with 0.1% acetic acid in each) at a flow rate of 1 mL/minute. Radioactivity was detected using an on-line flow detector and with UV absorption at 254 nm. Dilutions of the radiolabeled test substance were co-chromatographed with the unlabeled test substance. Typical retention times for the test substances were 5.8 and 10.8 minutes for Sample A and Sample B, respectively.

Details on distribution in tissues:

At the termination of this study, all tissue concentrations were low. With the exception of liver, kidneys, GI tract, blood and (in males only) bone, group mean tissue concentrations were less than 0.1 μg equiv/g. Mean concentrations of 0.40 μg equiv/g were present in the liver of male rats compared with 0.25 μg equiv/g in females. The amount of radioactivity present in the tissue and residual carcass of males and females was 0.76% and 0.87% respectively.

Details on excretion:

Over 120 hours, males and females excreted mean totals of 99% and 96% of administered radioactivity respectively. Urinary excretion accounted for mean totals of 21% and 34% for males and females respectively. The excretion of radioactivity was predominantly in faeces, accounting for mean totals of 78% and 61% of the dose for males and females respectively.

Metabolites identified:

no

Conclusions:

Following a single oral administration of 10 mg/kg of the [14C]-radiolabeled test substance to male and female rats, excretion was extensive and predominantly in faeces. Males excreted a greater proportion of the radioactivity in faeces and a lower proportion in urine than females. Tissue concentrations were low for both sexes.

Executive summary:

Five male and five female rats were each administered a single oral dose of 10 mg/kg of the [14C]-radiolabeled test substance. The study was carried out in two phases (phases A and B). Initially four rats of each sex were dosed (Sample A), followed by one rat of each sex (Sample B). The excretion of radioactivity in urine and faeces was monitored for 120 hours after dosing. After this period, the rats were killed and the remaining radioactivity was measured in blood, selected tissues and in the residual carcasses.

Over 120 hours, males and females excreted mean totals of 99% and 96% of administered radioactivity respectively. Urinary excretion accounted for mean totals of 21% and 34% for males and females respectively. The excretion of radioactivity was predominantly in faeces, accounting for mean totals of 78% and 61% of the dose for males and females respectively. Females also showed a higher percentage of the dose in the gastrointestinal tract contents taken at termination of the study, although for both sexes, 1% or less of the dose was present in the gastrointestinal tract contents.

 

At the termination of this study, all tissue concentrations were low. With the exception of liver, kidneys, GI tract, blood and (in males only) bone, group mean tissue concentrations were less than 0.1 μg equiv/g. Mean concentrations of 0.40 μg equiv/g were present in the liver of male rats compared with 0.25 μg equiv/g in females. The amount of radioactivity present in the tissue and residual carcass of males and females was 0.76% and 0.87%, respectively.

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:

test procedure in accordance with national standard methods

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:

99.8% purity

Radiolabelling:

yes

Species:

rat

Strain:

other: Alpk:Apf SD

Details on species / strain selection:

The rat was selected as a species recommended in the regulatory guidelines. The Alpk:APf SD strain of rat was selected because this strain was used during other toxicology studies with this test substance and since substantial background information is available for its use in the testing facility. The oral route of administration was used to represent a possible route of human exposure.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Not reported
- Weight at study initiation: 180-208 g
- Housing: The animals were acclimatized in stock cages for at least 5 days and then housed in the same cages for the duration of dosing with the unlabeled dose preparation. Immediately after the fourteenth unlabeled dose they were transferred to stainless steel metabolism cages, in which
they were individually housed and acclimatized for approximately one day prior to administration of the radiolabeled dose.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): At least 15 changes/hour, giving positive pressure with respect to the access corridor
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 600

Details on exposure:

Seven male and seven female rats were each given 14 consecutive daily oral doses of 10 mg/kg of the unlabeled test substance. Twenty four hours after the fourteenth dose five rats of each sex were each administered a single oral dose of 10 mg/kg of the [14C]-labeled test substance.

Duration and frequency of treatment / exposure:

Daily for 14 consecutive days with the unlabeled test substance and then one final dose on day 15 of the 14C-labeled test substance.

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Daily for 14 consecutive days with the unlabeled test substance and then one final dose of the 14C-labeled test substance on day 15.

No. of animals per sex per dose / concentration:

7 male and 7 female in the 14-day initial part of the study and then 5 males and 5 females in the metabolism/excretion part of the study

Control animals:

no

Positive control reference chemical:

No

Details on study design:

- Dose selection rationale: The dose level was selected to represent a 'no-effect' dose following a single oral administration.

Details on dosing and sampling:

The purity and concentration of the unlabeled test substance following formulation in the dosing vehicle was determined by HPLC using the following conditions: An Alltima Cl8, 5 μm, reverse phase column (150 x 4.6 mm, Alltech Associates Applied Science Ltd, Carnforth, Lancashire, UK) was eluted with acetonitrile:water (4:6 v/v) at a flow rate of 1 mL/minute. Acetic acid (0.1%) was added to both components of the mobile phase. The unlabeled test substance was detected with UV absorption at 254 nm. Concentrations of 1, 2.5, 5, 7.5 and 10 mg/mL of the unlabeled test substance in the dose vehicle were analyzed and a standard curve produced to determine achieved concentration. A typical retention time for the test substance was 10.35 minutes.

The stability of the unlabeled test substance following formulation in the dosing vehicle was determined by HPLC using the following conditions: An Alltima Cl8, 5 μm, reverse phase column (150 x 4.6 mm, Alltech Associates Applied Science Ltd, Carnforth, Lancashire, UK) was eluted with acetonitrile:water (4:6 v/v) at a flow rate of 1 mL/minute. Acetic acid (0.1%) was added to both components of the mobile phase. The unlabeled test substance was detected with UV absorption at 254 nm. Concentrations of 1, 2.5, 5, 7.5 and 10 mg/mL of the unlabeled test substance in the dose vehicle were analyzed and a standard curve produced to determine achieved concentration. A typical retention time for the test substance was 10.35 minutes.

The radiochemical concentration of the radiolabeled dose preparation was determined by taking triplicate weighed aliquots of the dose preparation and diluting each volumetrically in acetone. Triplicate aliquots of each dilution were taken for liquid scintillation counting.
The radiochemical purity of the radiolabeled test substance was determined, prior to and following formulation in the dose vehicle, by thin layer chromatography (TLC) using silica gel plates (60F2s4) in solvent systems 1-4:

1) Diethyl ether:dichloromethane (1:4 v/v)
2) Hexane:diethyl ether (1:9 v/v)
3) Hexane:ethyl acetate (1:1 v/v)
4) Chloroform:methanol:formic acid:water (75:25:4:4 v/v/v/v)

Dilutions of the [14C]-labeled test substance were co-chromatographed with the unlabeled test substance. Radioactivity on the TLC plates was measured using a Berthold Tracemaster linear analyser. Ther unlabeled test substance was visualized under UV light at absorption 254 nm. Typical Rf values for the test substance were 0.79, 0.74, 0.55 and 1 for systems 1, 2, 3 and 4 respectively.

The purity of the radiolabeled test substance prior to and following formulation in the dosing vehicle was determined by HPLC using the following conditions: An aliquot of the radiolabeled test substance was injected onto an Alltima C18, 5 μm, reverse phase column (150 x 4.6 mm, Alltech Associates Applied Science Ltd, Carnforth, Lancashire, UK). The column was eluted with water:acetonitrile (4:6 v/v) at a flow rate of 1 mL/minute and the main peak collected. Acetic acid (0.1%) was added to both components of the mobile phase. Radioactivity was detected using an on-line flow detector and with UV absorption at 254 nm. Dilutions of the radiolabeled test substance were co-chromatographed with the unlabeled test substance. A typical retention time for the test substance was 12 minutes.

Statistics:

No information was presented indicating whether statistics were used in this study.

Details on excretion:

Over 120 hours, males and females excreted mean totals of 97% and 96% of administered radioactivity respectively. Urinary excretion accounted for mean totals of 19% and 32% for males and females, respectively, with females showing a higher rate of excretion in urine than males throughout the study. The excretion of radioactivity was predominantly in the faeces, accounting for mean totals of 77% of the dose in males and 63% of the dose in females, with males showing a markedly higher rate of excretion between 12 and 24 hours than females. The rate of faecal excretion was higher in females between 48-120 hours. Furthermore, the amount of radioactivity excreted between 96 and 120 hours by 4 out of 5 females was greater than that excreted during the previous 24 hours (72-96 hours).

Metabolites identified:

no

Conclusions:

Following a single oral administration of 10 mg of the [14C]-labeled test substance to male and female rats previously administered fourteen daily oral doses of 10 mg/kg of the unlabeled test substance, excretion was extensive and predominantly in the faeces. Males excreted a greater proportion of the radioactivity in the faeces and a lower proportion in urine than females. Tissue concentrations were low for both sexes. Comparison of the results obtained in this study with those obtained following a single oral dose of 10 mg/kg of the [14C]-labeled test substance showed only minor differences in the excretion profiles and tissue concentrations with the exception of bone, with much lower concentrations in both males and females following the repeat dose regime.

Executive summary:

Seven male and seven female rats were each given 14 consecutive daily oral doses of 10 mg/kg of the unlabeled test substance. Twenty four hours after the fourteenth dose five rats of each sex were each administered a single oral dose of 10 mg/kg of the [14C]-labeled test substance. The excretion of radioactivity in urine and faeces was monitored for 120 hours after dosing. After this period, the rats were killed and the remaining radioactivity was measured in blood, selected tissues and in the residual carcasses.

Over 120 hours, males and females excreted mean totals of 97% and 96% of administered radioactivity respectively. Urinary excretion accounted for mean totals of 19% and 32% for males and females, respectively, with females showing a higher rate of excretion in urine than males throughout the study. The excretion of radioactivity was predominantly in the faeces, accounting for mean totals of 77% of the dose in males and 63% of the dose in females, with males showing a markedly higher rate of excretion between 12 and 24 hours than females. The rate of faecal excretion was higher in females between 48-120 hours. Furthermore, the amount of radioactivity excreted between 96 and 120 hours by 4 out of 5 females was greater than that excreted during the previous 24 hours (72-96 hours).

At termination of the study, the mean percentage of dose present in the gastrointestinal tract contents was 0.5% or less for both sexes. At the termination of this study, all tissue concentrations were low. With the exception of liver, kidneys, GI tract and blood, group mean tissue concentrations were 0.1 μg equiv/g or less. Mean concentrations of 0.47 μg equiv/g were present in liver of male rats compared with 0.26 μg equiv/g in females.

Reference 5

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:

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

99% purity for the technical, unlabeled material and >97% for the [14C]-phenylacrylate-labeled and the [14C]-pyridinyl-labeled test substance

Radiolabelling:

yes

Species:

rat

Strain:

other: Alpk:APf SD (Wistar-derived)

Details on species / strain selection:

The Alpk:APrSD strain of rat was used because of the substantial background available for this strain in the testing laboratory and this was the strain of rat used in toxicity studies on the test substance.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Not reported
- Weight at study initiation: 196-295 g (males); 188-275 g (females)
- Housing: The rats were housed in groups of the same sex in stock rat cages and acclimatized. Following this period, rats were transferred and housed individually in stainless steel metabolism cages where they were acclimatized for approximately 24 hours prior to dosing. Rats destined for bile duct cannulation experiments were transferred to individual glass metabolism cages where they were acclimatized for approximately 24 hours prior to surgery.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): At least 15 changes/hour, giving positive pressure with respect to the access corridor
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 600

Duration and frequency of treatment / exposure:

Single oral gavage dose

Dose / conc.:

100 mg/kg bw (total dose)

Remarks:

Either the phenylacrylate or pyridinyl 14C-labeled test substance was used

Dose / conc.:

10 mg/kg bw (total dose)

Remarks:

Either the phenylacrylate or pyridinyl 14C-labeled test substance was used

No. of animals per sex per dose / concentration:

16

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

No

Details on study design:

Dose selection rationale: The dose level selected for investigations in bile duct cannulated rats (100 mg/kg body weight) was compatabil with that used in other metabolism studies. Excreta from other studies were obtained from animals dosed with 10 or 100 mg/kg body weight.

Details on dosing and sampling:

Each rat was identified by tail numbering and was dosed by gavage using a syringe fitted with a dosing catheter. The amount of dose administered to each rat was determined by weighing the syringe and catheter assembly prior to and immediately after dosing. Immediately after dosing each rat was returned to its individual metabolism cage. A card giving the study number, animal number, dose level and other details of dosing was attached to each cage.

Statistics:

No

Details on absorption:

Following an oral dose of the test substance to the rat, 70-80% of the dose was absorbed.

Details on excretion:

Rats fitted with a biliary cannula excreted up to 72% of administered radioactivity in bile following an oral dose of 100 mg/kg of the [14C]-labeled test substance; male rats excreted up to 5% of the dose in urine whilst female rats excreted up to 24% by this route. Non-cannulated male and female rats excreted up to 26% and approximately 27% of the dose in urine, respectively, indicating enterohepatic recirculation.

Metabolites identified:

yes

Details on metabolites:

The test substance was extensively metabolized (42 metabolites, of which 34 were structurally identified). The major route of metabolism was ester hydrolysis and glucuronide conjugation, which was consistent between the sexes following both single and repeated doses at 10 mg/kg and following a single dose at 100 mg/kg. All metabolites which represented greater than 5% of the administered dose were identified, with the single exception of fecal metabolite A, which is considered to originate from bile, in which all significant metabolites were identified. This is endorsed by the identified metabolites in bile duct cannulated rats accounting for 91-101% of the administered dose. Identified metabolites in samples collected following single or repeat dose mass balance studies accounted for 73-83% of the dose.

Conclusions:

Following an oral dose of the test substance to the rat, 70-80% of the dose was absorbed, with the greatest proportion of the systemic dose eliminated via bile for subsequent excretion in feces.
The test substance was extensively metabolized, with the measurement in this study of 42 metabolites, of which 34 were structurally identified. The major route of metabolism was ester hydrolysis and glucuronide conjugation, which was consistent between the sexes following both single and repeated doses at 10 mg/kg and following a single dose at 100 mg/kg. All metabolites which represented greater than 5% of the administered dose were identified, with the single exception of fecal metabolite A, which is considered to originate from bile, in which all significant metabolites were identified. This is endorsed by the identified metabolites in bile duct cannulated rats accounting for 91-101%of the administered dose. Identified metabolites in samples collected following single or repeat dose mass balance studies accounted for 73-83% of the dose.

Executive summary:

The biotransformation of the test substance was investigated in rats administered oral doses of 10 mg or 100 mg/kg of the [14C]-phenylacrylate or pyridinyl labeled test substance. A single oral dose was administered to male and female rats; in addition, similar doses of phenylacrylate or pyridinyl labeled test substance were given to male and female rats that had been fitted with a bile duct cannula. The proportions of dose eliminated in bile, urine and feces were determined. Metabolites isolated from bile, urine and feces were characterized by mass spectroscopy and/or co-chromatography with reference standards. When required proton nuclear magnetic resonance (NMR) was used to give definitive structural information.  Metabolites present in excreta and bile from these animals were quantified by high performance liquid chromatography (HPLC) with radioactivity detection. Additional samples from the definitive excretion and tissue distribution studies were also analyzed. These urine samples and fecal extracts were from rats administered single oral doses of 10 or 100 mg/kg of the [14C]-phenylacrylate labeled or 10 mg/kg of the [14C]-phenylacrylate labeled test substance following fourteen consecutive daily doses of unlabeled test substance. The relative proportions of metabolites present in these samples were quantified by HPLC. Rats fitted with a biliary cannula excreted up to 72% of administered radioactivity in bile following an oral dose of 100 mg/kg of the [14C]-labeled test substance; male rats excreted up to 5% of the dose in urine whilst female rats excreted up to 24% by this route. Non-cannulated male and female rats excreted up to 26% and approximately 27% of the dose in urine respectively indicating enterohepatic recirculation. Several metabolic transformations occurred; ester hydrolysis, O-demethylation, hydroxylation and ring separation, together with additional metabolic reactions on the resulting metabolites producing at least 42 metabolites.

Reference 6

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:

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

Raw data notes for [phenyl(U)-14C]-test substance 100 mg/kg prep are absent from study record. Key information was collected iin DEBRA Lablogic program at time of conduct and dose confirmed by analysis, so the integrity of this group is not compromised.

GLP compliance:

yes

Specific details on test material used for the study:

The radiolabeled test material on the phenyl moiety had a purity of 99.4%.
The radiolabeled test material on the pyridine moiety had a purity of 98.2%.

Radiolabelling:

yes

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

Dose volume 4 mL/kg
c14 dose: 40 μCi/rat

Duration and frequency of treatment / exposure:

Single administration

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose / concentration:

4

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

No

Details on dosing and sampling:

Following dosing, rats were housed in glass metabolism units for collection of urine and faeces. Urine and faeces were collected on dry ice pre-dose, 0-6, 6-12, 12-24, 24-48, 48-72, 72-96, and 96-120 hours and analyzed for total 14C residues. Both the low and high dose groups were terminated at 120 hour.

Type:

excretion

Results:

The urinary excretion in male rats was 33.5% for the 10 mg/kg dose and 21.3% for the 100 mg/kg dose.

Type:

excretion

Results:

The urinary excretion in female rats was 39.6% for the 10 mg/kg dose and 41.0% for the 100 mg/kg dose.

Type:

excretion

Results:

The faecal excretion in male rats was 53.3% for the 10 mg/kg dose and 59.0% for the 100 mg/kg dose.

Type:

excretion

Results:

The faecal excretion in female rats was 49.4% for the 10 mg/kg dose and 40.0% for the 100 mg/kg dose.

Details on distribution in tissues:

The liver contained the highest percentage of residual 14C relative to all the other individual tissue samples by 120 hours after dose administration. The residual mean percentages in the liver were 0.26% for male rats and 0.15% for female rats and essentially identical for each sex regardless of dose level.

Details on excretion:

Urinary excretion was substantially complete by 48-72 hours after dose administration. The range of mean values for urinary excretion was 21.3% to 41.0%. The range of faecal excretion was 40.0% to 59.0%.

Metabolites identified:

no

Executive summary:

The tissue distribution of the 14C-phenyl and 14C-pyridine-labeled test substance was investigated in male and female Sprague-Dawley Crl:CD(SD) rats. The radiolabeled test substance was administered to male and female rats by single oral gavage. Based on the plasma pharmacokinetic experiments which showed similar results when the [phenyl(U)-14C] and [pyridine-3-14C] radiolabels were administered separately, tissue distribution experiments were conducted at 2 dose levels by combining the labels in a 1:1 μCi ratio. Tissue 14C residues were measured at 1, 24 and 120 hours after administration of the low (10 mg/kg) dose and 24, 48 and 120 hours after administration of the high (100 mg/kg) dose. The 120-hour sampling time included measurement of material balance to insure consistency with previous study results.

The tissue distribution results demonstrated uptake and extensive systemic distribution of the 14C-test substance. No major sex differences were observed in the overall distribution. The majority of the administered dose was associated with the gastrointestinal tract contents followed by the gastrointestinal tract tissue and liver. The mean values for total body burden were 65.9% and 70.1% of the dose at Tmax (1 h); these declined to 1.7% and 1.9% by 120 hours after low (10 mg/kg) dose administration in male and female rats, respectively. At the high (100 mg/kg) dose, the total body burden was 52.5% and 48.1% at Tmax (24 h) and declined to 4.2% and 2.0% of the dose by 120 hours after dosing of male and female rats, respectively. At terminal collection (120 hours), the percentages in the liver were quantitatively the same at both dose levels with mean values of 0.26% for male rats and 0.15% for female rats, respectively. Tissue concentrations at the terminal (120 h) collection increased in direct proportion with dose. Excluding the gastrointestinal tract tissue and contents, the other tissues showed a low potential

for accumulation based on the tissue:plasma ratios that ranged from 0.06:1 (brain) to 5:1 (liver) at all 3 sample times, for both sexes, and both dose levels. Comparison of plasma and liver 14C residue concentrations from the current study with the plasma time course data showed reasonably good agreement in the magnitude of 14C concentrations and pattern of decline. The results in conjunction with biliary elimination data previously reported provide evidence for enterohepatic recirculation playing a role in the overall kinetic behaviour of the test substance in the rat.

 The rate and extent of urinary and faecal excretion and material balance at 120 hours after dose administration were generally consistent with previously reported data. Urinary excretion was substantially complete by 48-72 hours after dose administration. The range of mean values for urinary excretion in the current study (21.3% to 41.0%) was similar and possibly slightly higher than the range from the previous studies (17.8% to 33.8%). The range of faecal excretion from the previous studies (59.0% to 77.8%) appeared to be slightly higher than that measured in the current study (40.0% to 59.0%). Radioactive 14C residues in tissues (including the gastrointestinal tract contents) ranged from 1.07% to 1.71% in the previous studies compared with 1.69% to 4.25% in the current study. These findings suggest slightly higher absorption in the current study than the previous studies. Material balance for the previous and current studies through terminal (120 hour) collection ranged from 90.9% to 99.3%.

Additional information

The absorption, distribution, metabolism and excretion of [14C]test substance was investigated in rats after single low (10 mg/kg bw) and high (100 mg/kg bw) dose administration and after multiple treatments at low doses. The test substance was well absorbed (>75%) in rats following oral administration. Most of the administered dose was eliminated via bile for subsequent excretion in faeces. In both sexes, excretion was both rapid and extensive with 99 and 96% of the administered dose excreted over 120 hours in males and females, respectively. Elimination in expired air was negligible. Tissue clearance was rapid with <2% remaining in tissues after 120 hours after low and high dose administration. Test substance was extensively metabolised; 42 metabolites were found of which 34 were structurally identified; 73-83% of the dose was accounted for by the identified metabolites. The predominant biotransformation reaction was ester hydrolysis and glucuronide conjugation. O-demethylation was also a major route of metabolism with subsequent oxidation or reduction and conjugation reactions. Metabolism was consistent between the sexes, between the low and high dose administration, and between single and repeated oral dosing.