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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:
EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Test Guidelines for Agricultural Chemicals 2-3-1 Notification 12-Nousan-8147
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD®(SD)IGS BR
Details on species / strain selection:
The Sprague-Dawley rat was chosen for this study because of the extensive experience with this strain and its suitability with respect to longevity, sensitivity, and low incidence of spontaneous diseases. Furthermore, the Sprague-Dawley rat has been used previously for toxicity testing of this chemical.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, U.S.A.
- Age at study initiation: 8-11 weeks old (non-cannulated animals were approximately 8 weeks old, except for the high (200 mg/kg bw) dose group administered which had animals that were approximately 9 weeks old. Cannulated animals were approximately 11 weeks old for the pharmacokinetic and biliary elimination experiments).
- Weight at study initiation: 205 (±10) g to 345 (±20) g for male rats and 159 (±6) g to 237 (±20) g for female rats
- Housing: During the pretest period, animals were group housed (non-cannulated) or housed individually (cannulated) in solid bottom caging with bedding and nestlets. Each cage rack contained animals of either sex. Animals were moved to glass metabolism units for the in-life phase of the study. Animals for the Tmax collection times of the tissue distribution experiment were placed in solid bottom cages with a wire metal grate added to accommodate the short collection interval during the in-life phase.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): tap water
- Acclimation period: The animals were quarantined for at least 6 days with the exception of cannulated rats, which were quarantined for at least 3 days. Other experiment specific exceptions included rats for the low dose distribution experiment which were quarantined for 5 days. Male rats for the low dose material balance experiment were quarantined for 4 days and female rats for this dose group were quarantined for 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30-70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): Animal rooms were artificially illuminated (fluorescent light) on an approximate 12 hour light/dark cycle.

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
other: 0.1% Tween 80 in 0.5% methylcellulose
Duration and frequency of treatment / exposure:
Single dose
Dose / conc.:
10 mg/kg bw (total dose)
Remarks:
[pyridine-2,6-14C]
Dose / conc.:
200 mg/kg bw (total dose)
Remarks:
[pyridine-2,6-14C]
Dose / conc.:
10 mg/kg bw (total dose)
Remarks:
[fused pyrimidine-3-14C]
Dose / conc.:
200 mg/kg bw (total dose)
Remarks:
[fused pyrimidine-3-14C]
Dose / conc.:
10 mg/kg bw (total dose)
Remarks:
[methylene-14C]
Dose / conc.:
200 mg/kg bw (total dose)
Remarks:
[methylene-14C]
No. of animals per sex per dose:
1 male and 1 female for the vehicle in the Pilot Excretion, Material Balance and Tissue Distribution, Pharmacokinetic, Biliary Elimination, and Tissue Distribution studies and 4 males and 4 females in the Main Pilot Excretion, Material Balance and Tissue Distribution, Pharmacokinetic, Biliary Elimination, and Tissue Distribution studies
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected to bracket the 2 lowest mean daily intake doses in a 28-day feeding toxicity study and the 2 middle doses in a 13-week feeding study in rats
- Rationale for animal assignment (if not random): Animals were selected for use on study based on adequate body weight gain and freedom from any clinical signs of disease or injury.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, tissues, carcass, cage washes, exhaled breath, plasma, red blood cells, bile, and residual feed
- Time and frequency of sampling: Urine and feces samples were collected pre-dose and at 0-6, 6-12, 12-24, 24-48, 48-72, 72-96, 96-120, 120-144, and 144-168 hour intervals
- Other:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Whole blood, urine, faeces, tissues, carcass, cage washes, and residual feed
- Time and frequency of sampling: 2 hours post-dose, and plasma was submitted for metabolite profiling
- From how many animals: 4 males and 4 females; aliquots of homogenized feces samples were pooled across the four animals per sex per dose group for the 0-24 and 24-48 hour collection intervals, respectively.
- Method type(s) for identification: Metabolite identification was conducted by HPLC/MS chromatography and a combination of mass spectral fragmentation pattern, accurate mass of the molecular ion, and retention time comparison with available reference standards
- Limits of detection and quantification: the limit of detection (LOD) and limit of quantitation (LOQ) values were set to 2 and 3 times the background mean, respectively.
Statistics:
Group data are represented as a mean ± SD. Only the values obtained were used in the mean calculation. Individual animal values reported as
All calculations were carried out with attention to maintaining an adequate number of significant figures so as not to inadvertently misrepresent results. Tables and appendices presented in the report were computer generated and values were rounded appropriately for inclusion in this report. Consequently, the application of manual calculation to report values may in some instances yield a minor variation. These occurrences should not be construed as adversely affecting the integrity or interpretation of the data.
Preliminary studies:
For the pilot excretion experiments, the percentage of administered dose recovered from all groups by 7 day after dose administration ranged from 89.7 to 114% for all three labels. The majority of the administered dose was recovered in the urine (38.1 to 49.4%) and feces (40.4 to 57.4%) with no apparent difference between sexes. A key finding was that no appreciable radioactivity from any of the three radiolabels was eliminated in exhaled breath.
Type:
absorption
Results:
Very rapid absorption. Absorption was slightly reduced at the high (200 mg/kg bw) dose with a mean of 63 (±12)% that was consistent with slight saturation of absorption.
Type:
distribution
Results:
Mean distribution was 43.3-52.6% in feces, 39.9-47.8% in urine, and 0.364-0.586% in tissues (low dose) and 56.8-68.8% in feces, 25.0-35.3% in urine and 0.116- 0.415% in tissues (high dose).
Type:
metabolism
Results:
Metabolism was similar across dose, sex, and the three radiolabels. The proposed metabolic pathway included a series of hydroxylation, oxidation, hydrolysis, decarboxylation, and rearrangement reactions at various locations throughout the molecule.
Type:
excretion
Results:
A significant portion was excreted in urine and feces as the intact test substance. The high percentage for urinary excretion of the intact test substance can be attributed to its high water solubility (0.23 g/L at 20°C).
Details on absorption:
Absorption was rapid. Mean absorption half-lives estimated by compartmental analysis ranged from 0.051-0.14 hour (3-8 minutes) and 0.28-1.1 hours (17-66 min) after low and high dose administration, respectively. Mean Tmax values at the low dose for all three labels ranged from 0.3 to 0.8 hours. Tmax at the high dose ranged from 1.8 to 2.5 hours for all three radiolabels.
Details on distribution in tissues:
The mean distribution of radioactivity for rats administered the low (10 mg/kg bw) dose for all three radiolabels was 43.3-52.6% in feces, 39.9-47.8% in urine, and 0.364-0.586% in tissues. For rats administered the high (200 mg/kg bw) dose of each radiolabel, the mean distribution was 56.8-68.8% in feces, 25.0-35.3% in urine and 0.116- 0.415% in tissues.

The percentages and concentration values of quantifiable [14C]residues in all collected tissues were very low. Mean tissue concentrations, with the exception of the GI tract tissue, were less than 22 μg equivalents/g at Tmax after low dose administration for all three radiolabels. The majority of the dose at Tmax was associated with the gastrointestinal tract contents. By 168 hours, the tissue concentrations had declined to 0.09% and 0.03% after low and high dose administration, respectively. No major radiolabel or sex differences were observed in the overall distribution. Tissue:plasma concentration ratios indicated no potential for accumulation.
Details on excretion:
The majority of the dose was excreted in the feces and urine by 24 hours after dose administration. The mean distribution of radioactivity for rats administered the low (10 mg/kg bw) dose for all three radiolabels was 43.3-52.6% in feces, 39.9-47.8% in urine, and 0.364-0.586% in tissues. For rats administered the high (200 mg/kg bw) dose of each radiolabel, the mean distribution was 56.8-68.8% in feces, 25.0-35.3% in urine and 0.116- 0.415% in tissues.
Key result
Toxicokinetic parameters:
half-life 1st: 1.4-1.8 hours
Remarks:
10 mg/kg dose
Key result
Toxicokinetic parameters:
half-life 1st: 3.8-5.4 hourshours
Remarks:
200 mg/kg dose
Key result
Toxicokinetic parameters:
Tmax: 0.3-0.8 hours
Remarks:
10 mg/kg dose
Key result
Toxicokinetic parameters:
Tmax: 1.8-2.5 hours
Remarks:
200 mg/kg dose
Key result
Toxicokinetic parameters:
AUC: 83.2% (males) and 93.6% (females)
Remarks:
10 mg/kg dose and at 0-48 hours
Key result
Toxicokinetic parameters:
AUC: 88.9% (males) and 93.9% (females)
Remarks:
200 mg/kg dose and at 0-48 hours
Key result
Toxicokinetic parameters:
AUC: 10.8% (males) and 3.86% (females)
Remarks:
10 mg/kg dose and at 48-120 hours
Key result
Toxicokinetic parameters:
AUC: 6.82% (males) and 4.10% (females)
Remarks:
200 mg/kg dose and at 48-120 hours
Metabolites identified:
yes
Details on metabolites:
The metabolism of the test substance was similar across dose, sex, and the three radiolabels. The proposed metabolic pathway included a series of hydroxylation, oxidation, hydrolysis, decarboxylation, and rearrangement reactions at various locations throughout the molecule. The most significant reaction was hydroxylation of the trifluoromethyl phenyl ring to form IN-R6U70, which was found in feces (14-27%) and to a lesser extent in urine (0.7-2.2%). Sulfation and glucuronidation of IN-R6U70 also was observed. Other minor proposed metabolic reactions included hydroxylation (IN-R6U71 and four isomers of OH-RAB55), N-oxidation (IN-R3Z91), oxidative hydrolysis (IN-RPD47 and IN-Y2186), sequential oxidative hydrolysis, decarboxylation, and rearrangement (IN-SBV06 and IN-RUB93), and hydrolytic cleavage (IN-RPA19). Additional proposed reactions on the metabolites of DPX-RAB55 included hydrolytic cleavage of IN-R6U70, IN-R6U71 and IN-R6U72 (IN-RPA19), hydrolytic defluorination of IN-R6U70 (IN-R6U72), amide hydrolysis of IN-SBV06 or IN-RUB93 (IN-RUA92), hydroxylation of IN-SBV06 and IN-RUB93, and glucuronide metabolites of OH-RAB55, IN-R6U71, and OH-RUB93.
Conclusions:
The test substance was readily excreted as the parent molecule representing 43-63% of the administered dose recovered in urine and feces. Approximately 24-43% of the administered dose was recovered in urine and feces as metabolized residues. The proposed metabolic pathway included a series of hydroxylation, oxidation, hydrolysis, decarboxylation, and rearrangement reactions at various locations throughout the molecule. The most significant reaction was hydroxylation of the trifluoromethyl phenyl ring to form IN-R6U70, which was found in feces (14-27%) and to a lesser extent in urine (0.7-2.2%). Sulfation and glucuronidation of IN-R6U70 also was observed. Other minor metabolites included IN-R6U71, IN-R3Z91, IN-RPD47, IN-SBV06, IN-RUB93, IN-RPA19, IN-R6U72, IN-Y2186, and IN-RUA92. OH-SBV06, OH-RUB93, four isomers of OH-RAB55, and glucuronide metabolites of OH-RAB55, IN-R6U71, and OH-RUB93 were proposed.
Executive summary:

The absorption, distribution, metabolism, and excretion of the test substance was studied in male and female rats. Experiments were performed to understand the disposition and material balance of total 14C residues among tissues, and excreta (including exhaled breath), pharmacokinetic behavior of radioactive residues in plasma and red blood cells, measurement of absorption thru elimination of 14C residues in bile, and the percentage and concentration of 14C residues in tissues at selected times after dosing. The experiments were performed using three radiolabeled forms: [pyridine-2,6-14C], [fused pyrimidine-3-14C], or [methylene-14C], each mixed with the non-radiolabeled test material (OECD Test Guideline 417).

For the pilot excretion experiments, the percentage of administered dose recovered from all groups by day 7 after dose administration ranged from 89.7 to 114% for all three labels. The majority of the administered dose was recovered in the urine (38.1 to 49.4%) and feces (40.4 to 57.4%) with no apparent difference between sexes. A key finding was that no appreciable radioactivity from any of the three radiolabels was eliminated in exhaled breath.

 

In the definitive material balance experiments, the mean percentage of administered dose recovered from all groups by 168 hours after dose administration ranged from 89.0% to 102%. The majority of the dose was excreted in the feces and urine by 24 hours after dose administration. The mean distribution of radioactivity for rats administered the low (10 mg/kg bw) dose for all three radiolabels was 43.3-52.6% in feces, 39.9-47.8% in urine, and 0.364-0.586% in tissues. For rats administered the high (200 mg/kg bw) dose of each radiolabel, the mean distribution was 56.8-68.8% in feces, 25.0-35.3% in urine and 0.116- 0.415% in tissues. The overall material balance was similar across sexes and radiolabels. The tissue burden and urinary excretion at the high dose were respectively 1.4- to 3.1-fold and 1.3- to 1.7-fold lower than the low dose. Fecal excretion increased by 1.1- to 1.4- fold as the administered dose was increased from 10 to 200 mg/kg bw. These results indicated a slight saturation of absorption after high dose administration.

 

The pharmacokinetics of 14C residues in plasma following oral gavage administration showed similar biphasic elimination kinetics among the three radiolabels with very rapid absorption, rapid distribution, and no significant sex differences. Cmax and AUC values were also similar across the respective dose groups. The majority of the dose was eliminated by 24-48 hours based on the rapid tissue distribution half-lives (1.4-5.4 hours) and AUC partial area percentages (82-96%). The remaining residues (4-18%) were eliminated with an average elimination half-life of 46 (±7) hours (n=40 rats). Group mean elimination half-lives at both dose levels ranged from 35 to 54 hours, except for the high dose [methylene-14C] mean elimination half-lives of 12-14 hours. The individual animal values for these means were not included in the overall mean value of 46 hours. The pharmacokinetic results were consistent with the rate and extent of excretion in urine and feces from the material balance experiments. Plasma and red blood cell 14C residue concentrations were similar throughout all sampling times indicating the absence of binding in red blood cells.

 

The biliary elimination results showed that the mean percent absorption at the low (10 mg/kg bw) dose was 84 (±5)% across both sexes and three radiolabels. Absorption was slightly reduced at the high (200 mg/kg bw) dose with a mean of 63 (±12)% that was consistent with slight saturation of absorption.

 

From the tissue distribution experiments, systemic uptake and distribution was evident based on the presence of quantifiable 14C residues in all the collected tissues. However, the percentages and concentration values were very low. Mean tissue concentrations, with the exception of the GI tract tissue, were less than 22 μg equivalents/g at Tmax after low dose administration for all three radiolabels. The majority of the dose at Tmax was associated with the gastrointestinal tract contents. By 168 hours, the tissue concentrations had declined substantially by 2 to 4 orders of magnitude with concentrations ranging between 0.001 to 0.035 μg equivalents/g, and a number of tissues had concentrations that were <LOQ. A similar concentration distribution pattern was observed in tissues of rats administered the high (200 mg/kg bw) dose of each radiolabel. By 168 hours after dose administration, the mean percentages of the dose in each tissue declined substantially, ranging from <LOQ to 0.09% and <LOQ to 0.03% after low and high dose administration, respectively. Slightly lower percentages of 14C recovery in tissues after high (200 mg/kg bw) dose administration was consistent with a reduction in absorption observed in the biliary elimination experiments and increased recovery in feces observed in the material balance experiments. No major radiolabel or sex differences were observed in the overall distribution. Tissue:plasma concentration ratios indicated no potential for accumulation.

 

A significant portion of the low and high dose was excreted in urine (20-41%) and feces (12-37%) as the intact test substance. The high percentage for urinary excretion of the unchanged test substance can be attributed to its high water solubility (0.23 g/L at 20°C). The parent test substance was also the major component in plasma. The metabolism of the test substance was similar across dose, sex, and the three radiolabels. The proposed metabolic pathway included a series of hydroxylation, oxidation, hydrolysis, decarboxylation, and rearrangement reactions at various locations throughout the molecule. The most significant reaction was hydroxylation of the trifluoromethyl phenyl ring to form IN-R6U70, which was found in feces (14-27%) and to a lesser extent in urine (0.7-2.2%). Sulfation and glucuronidation of IN-R6U70 also was observed. Other minor proposed metabolic reactions included hydroxylation (IN-R6U71 and four isomers of OH-RAB55), N-oxidation (IN-R3Z91), oxidative hydrolysis (IN-RPD47 and IN-Y2186), sequential oxidative hydrolysis, decarboxylation, and rearrangement (IN-SBV06 and IN-RUB93), and hydrolytic cleavage (IN-RPA19). Additional proposed reactions on the metabolites of DPX-RAB55 included hydrolytic cleavage of IN-R6U70, IN-R6U71 and IN-R6U72 (IN-RPA19), hydrolytic defluorination of IN-R6U70 (IN-R6U72), amide hydrolysis of IN-SBV06 or IN-RUB93 (IN-RUA92), hydroxylation of IN-SBV06 and IN-RUB93, and glucuronide metabolites of OH-RAB55, IN-R6U71, and OH-RUB93.

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:
absorption
distribution
excretion
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Test Guidelines for Agricultural Chemicals 2-3-1 Notification 12-Nousan-8147
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
[pyridine-2,6-14C]
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD®(SD)
Details on species / strain selection:
The Sprague-Dawley rat was chosen for this study because of the extensive experience with this strain and its suitability with respect to longevity, sensitivity, and low incidence of spontaneous diseases. Furthermore, the Sprague-Dawley rat has been used previously for toxicokinetic and toxicity testing of this chemical.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, North Carolina, U.S.A.)
- Age at study initiation: 8 weeks
- Weight at study initiation: 217 ± 15 g
- Housing: During the pretest period, animals were group housed in solid bottom caging with bedding and Nestlets. Animals were moved to glass metabolism units for the in-life phase of the study.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30-70%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
other: 0.1% Tween 80 in 0.5% methylcellulose
Duration and frequency of treatment / exposure:
Daily for 14 days
Dose / conc.:
10 mg/kg bw/day
No. of animals per sex per dose:
1 female rat in the control group and 16 female rats in the study group
Control animals:
yes, concurrent vehicle
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum or other tissues, cage washes, feed residue, and carcass
- Time and frequency of sampling: Urine and faeces at 24-hour intervals
- Other:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, tissues, cage washes, bile
- Time and frequency of sampling:
- From how many animals: (samples pooled or not)
- Method type(s) for identification (e.g. GC-FID, GC-MS, HPLC-DAD, HPLC-MS-MS, HPLC-UV, Liquid scintillation counting, NMR, TLC)
- Limits of detection and quantification:
- Other:
TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum or other tissues, cage washes, feed residue, and carcass
- Time and frequency of sampling: urine and faeces at 24 hour intervals and blood, plasma, serum or other tissues, cage washes, feed residue, and carcass at the end of the 14-day study
- Other: 14C residues in selected tissues (whole blood, plasma, red blood cells, fat, kidney, liver, muscle and uterus) on days 13(+0.5 hr), 13(+8 hr), 14 and 18 for determination of elimination from tissues after dosing
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, plasma, and faeces
- Time and frequency of sampling: urine and feces collected for 24-hour intervals after the first, seventh, and last (fourteenth) day of dose administration
- From how many animals: 4
- Method type(s) for identification: HPLC-MS and LSC
- Limits of detection and quantification: LOD = 2 times and LOQ = 3 times the background mean
Statistics:
Group data were represented as a mean ± SD. Only the values obtained were used in the mean calculation. Individual animal values reported as All calculations were carried out with attention to maintaining an adequate number of significant figures so as not to inadvertently misrepresent results. Tables and appendices presented in the report were computer generated and values were rounded appropriately for inclusion in the report. Consequently, the application of manual calculation to report values may in some instances yield a minor variation. These occurrences should not be construed as adversely affecting the integrity or interpretation of the data.
Type:
distribution
Results:
The highest concentrations were observed in the liver 0.5 hours after the last dose (11.1 μg eq./g). Liver concentrations declined to 1.44, 0.382, and 0.117 μg eq./g at 8, 24 and 120 hours, respectively. Other tissues followed a similar pattern.
Type:
excretion
Results:
During dosing, the mean daily excretion in feces ranged from 42.9 to 49.0% of the dose; mean 46.4%. Urine excretion ranged from 36.9 to 38.8% of the dose; mean 38.7%. Total excretion in feces and urine was 85.1%.
Type:
metabolism
Results:
The intact test substance and 6 metabolites (IN-R6U70, IN-R6U72, IN-R3Z91, IN-RPD47, IN-RPA19, OH-RAB55 isomer)were identified among the feces, urine and plasma.
Type:
other: Material balance
Results:
The material balance group had a total mean recovery of 90.7(±4.4)%. The mean recovery of 14C residues in tissues including the carcass accounted 0.47% of the dose.
Details on excretion:
Retention of 14C residues in tissues was low (0.47%). A significant portion of the administered dose (52-60%) was excreted in the urine and feces as intact test substance. The unmetabolized test substance represented 95% of the 14C residue concentration in plasma sampled 30 minutes after administration of the last of the 14 daily doses.
Key result
Toxicokinetic parameters:
Cmax:
Remarks:
Concentrations (μg eq./g) mean values at 312.5 hour (0.5 hour after the last of dose) ranked as follows: liver (11.1) > kidney (9.91) > plasma (3.20) ~ uterus (3.17) > whole blood (2.94) ~ red blood cells (2.69) > muscle (2.40) > fat (1.20).
Key result
Toxicokinetic parameters:
half-life 1st: Plasma
Remarks:
48 hours
Key result
Toxicokinetic parameters:
half-life 1st: Red Blood Cells
Remarks:
165 hours
Key result
Toxicokinetic parameters:
half-life 1st: Blood
Remarks:
86 hours
Key result
Toxicokinetic parameters:
half-life 1st: Fat
Remarks:
8 hours
Key result
Toxicokinetic parameters:
half-life 1st: Liver
Remarks:
56 hours
Key result
Toxicokinetic parameters:
half-life 1st: Kidney
Remarks:
68 hours
Key result
Toxicokinetic parameters:
half-life 1st: Muscle
Remarks:
208 hours
Key result
Toxicokinetic parameters:
half-life 1st: Uterus
Remarks:
83 hours
Metabolites identified:
yes
Details on metabolites:
The test substance was subject to a series of hydroxylation, oxidation, hydrolysis, decarboxylation, and rearrangement reactions at various locations throughout the molecule. Six metabolites were observed (IN-R6U70, IN-R6U72, IN-R3Z91, IN-RPA19, IN-RPD47, and one OH-RAB55 isomer). The profile of metabolites was similar across the 0-24, 144-168 and 312-336 hour collection intervals indicating metabolism was not altered by multiple dose administration.
Conclusions:
The overall mean material balance was 90.7% of the cumulative dose administered. The dose was excreted in the feces (46.4%) and urine (38.7%) in similar proportions. Retention of 14C residues in tissues was low (0.47%).
Executive summary:

The kinetics of the radiolabeled test substance was determined in rats during and following multiple oral dose administration [10 mg/kg day for 14 days] (OECD Test Guideline 417). The highest concentrations were observed in the liver 0.5 hours after the last of 14 doses (11.1 μg equivalents/g). Concentrations in liver declined to 1.44, 0.382, and 0.117 μg equivalents/g at 8, 24 and 120 hours after the last dose, respectively. The other tissues followed a similar pattern of rapid decline of peak concentrations. Cmax concentrations after the last 14th dose declined very rapidly from 0.5 hours through 8 hours until 24 hours, at which time the rate of decline showed biphasic behavior and a slower rate of decline from 24 to 120 hours. Half-lives were calculated using a two-point slope calculation for the slower terminal elimination phase. For plasma the observed terminal elimination half-life was 48 hours. The terminal elimination half-lives in selected tissues (estimated using a two-point slope calculation for the slower terminal elimination phase) were fat (8 hours) << plasma (48 hours) < liver (56 hours) < kidney (68 hours) < uterus (83 hours) ~ blood (86 hours) < red blood cells (165 hours) and muscle (208 hours). The observed range of terminal half-lives reflects variability among tissues for retention of very low amounts of 14C residues. Tissue:plasma ratios were highest for liver and kidney which had values that ranged from 2:1 to 5:1. Tissue burdens were very low based on peak percentages ranging from 0.00548% (uterus) to 0.733% (muscle) of the administered dose 0.5 hours after the last dose. The tissue 14C residues subsequently declined to <LOQ (fat), and 0.00006% (uterus) to 0.0085% (muscle) of the total administered dose by 120 hours after the last dose. Tissue:plasma ratio and the percent of dose results indicated very low potential for accumulation of 14C residues. No accumulation of 14C residues occurred in fat or muscle, tissues of common concern for residue retention. The overall mean material balance was 90.7% of the cumulative dose administered. The dose was excreted in the feces (46.4%) and urine (38.7%) in similar proportions. Retention of 14C residues in tissues was low (0.47%). The test substance was subject to a series of hydroxylation, oxidation, hydrolysis, decarboxylation, and rearrangement reactions at various locations throughout the molecule. Six metabolites were observed (IN-R6U70, IN-R6U72, IN-R3Z91, IN-RPA19, IN-RPD47, and one OH-RAB55 isomer). The profile of metabolites was similar across the 0-24, 144-168 and 312-336 hour collection intervals indicating metabolism was not altered by multiple dose administration. A significant portion of the administered dose (52-60%) was excreted in the urine and feces as intact test substance. The unmetabolized test substance represented 95% of the 14C residue concentration in plasma sampled 30 minutes after administration of the last of the 14 daily doses. The hydroxylated metabolite IN-R6U70 was a major component found in feces and to a lesser extent in urine.

Description of key information

The material balance, pattern of distribution, and profile of metabolites were consistent between the single dose and multiple dose studies. Tissue-to-plasma ratios and low levels of total radioactive residues in tissues showed no significant potential for bioaccumulation of the test substance and its metabolites. The dose was principally excreted in the faeces and urine and retention of [14C]residues in tissues including the carcass was low. The pharmacokinetics of [14C]residues in plasma following single and multiple oral gavage administration showed biphasic elimination kinetics with very rapid absorption, rapid distribution, and no significant sex differences. Metabolism was extensive and characterised by several identified components in faeces, urine and plasma.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Absorption: Biliary elimination results showed that absorption for [14C]triflumezopyrim at the low (10 mg/kg bw) dose was ca.84% based on the sum of urine, bile, carcass, whole blood, and G.I. tract contents. Absorption was slightly reduced at the high (200mg/kg bw) dose at ca.63% due to slight saturation. Mean Tmax values ranged from 0.3 to 0.8 hours at the low dose and 1.8 to 2.5 hours at the high dose.

 

Distribution: Quantifiable 14Cresidues in all collected tissues (percentages and concentration values) were low. Mean tissue concentrations, with the exception of the GI tract tissue, were less than 22 µg equivalents/g at Tmax after low dose administration. By 168 hours, the tissue concentrations had declined to 0.035µg equivalents/g. A similar distribution pattern was observed in tissues of rats administered the high (200 mg/kg bw) dose. Tissue:plasma concentration ratios indicated no potential for accumulation. By 168 hours, the tissue concentrations had declined to 0.09% and 0.03% after low and high dose administration, respectively. No major radiolabel or sex differences were observed in the overall distribution.

 

Potential for accumulation: The low percentage and concentration values in tissues indicate every low potential for accumulation.

 

Rate and extent of excretion: The mean percentage of administered dose recovered from all groups by 168 hours after single dose administration ranged from 89.0 to 102%. Recovery ranged from 43.3 to 52.6% in faeces and 39.9 to 47.8% in urine across all low (10 mg/kg bw) dose treatment groups. The recovery in faeces increased slightly after high (200 mg/kg bw) dose administration (56.4 to 68.8%) and declined slightly in urine (25.0 to 35.3%). The majority of excretion occurred in the first 24 hours and was substantially complete by 48 hours after dose administration. Essentially no excretion occurred by exhalation.

 

The pharmacokinetics of 14Cresidues in plasma following single oral gavage administration showed biphasic elimination kinetics with very rapid absorption, rapid distribution, and no significant sex differences. The majority of the dose was eliminated by 24 -48 hours with the remaining residues (4 -18%) eliminated with an average elimination half-life of 46 hours. Mean elimination half-lives at both dose levels ranged from 35 to 54 hours,with the exception of the high dose [methylene-14C]-label (12 -14 hours).

 

Metabolic profile: The metabolism of the test substance was similar across dose, sex, and the three radiolabels. The proposed metabolic pathway included a series of hydroxylation, oxidation, hydrolysis, decarboxylation, and rearrangement reactions at various locations throughout the molecule. A significant portion of the low and high dose was excreted in urine (20 -41%) and faeces (12 -37%) as intact triflumezopyrim.  The most significant reaction was hydroxylation of the triflumezopyrim trifluoromethylphenyl ring to form IN-R6U70, which was found in faeces (14 -27%) and to a lesser extent in urine (0.7-2.2%). Sulfation and glucuronidation of IN-R6U70 was also observed. Minor proposed metabolic reactions included hydroxylation (IN-R6U71 and four isomers of OH-RAB55), N-oxidation (IN-R3Z91), oxidative hydrolysis (IN-RPD47and IN-Y2186), a series of oxidative hydrolysis, decarboxylation, rearrangement reactions yielding IN-SBV06 and IN-RUB93, and hydrolytic cleavage (IN-RPA19). Proposed reactions on metabolites of triflumezopyrim included hydrolytic defluorination of IN-R6U70 (IN-R6U72), amide hydrolysis of IN-SBV06 o rIN-RUB93 (IN-RUA92), hydroxylation of IN-SBV06 and IN-RUB93, and glucuronidation of OH-RAB55, IN-R6U71, and OH-RUB93.