5,7-dichloro-4-(4-fluorophenoxy)quinoline;quinoxyfen (ISO)

Endpoint summary

Link to relevant study record(s)

Reference

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:

metabolism

other: Pharmocokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Method No. 87/302

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Non-radiolabelled test substance: XDE-795
Substance ID: TSN 100010
Lot Number: DECO-104-116
Purity: 99%

Radiolabelled test substance: 14C-XDE-79 uniformly labelled in the phenyl ring and in the 2-position of the quinoline ring
Reference ID: GHD 3195-7 for phenyl labelled and GHD-3058-19 for quinoline labelled test substance
Radiochemical Purity: 98.5% for phenyl labelled and >99% for quinoline labelled test substance

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

The Fischer 344 rat was used since the rat is the preferred species for pharmacokinetic and metabolism studies and this strain has been used in other toxicity studies with test substance.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding laboratory, Kingston, New York
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Male: 180-250 grams; Female: 130-180 grams
- Housing: Glass Roth-type metabolism cages.
- Diet: Purina certified Rodent Chow (#5002), ad libitum, except that food was withdrawn approximately 10-17 hr prior to dosing with the radiotracer and returned approximately 4 hr postdosing.
- Water: Municipal drinking water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
The rooms in which the animals were housed had a 12-hr photocycle and were designed to maintain adequate environmental conditions concerning temperature and relative humidity for rodents.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% METHOCEL cellulose ethers

Details on exposure:

PREPARATION OF DOSING SOLUTIONS CONTAINING RADIOLABELLED TEST SUBSTANCE:
The 14C-labelled test substance dose solutions were prepared as aqueous suspensions in approximately 0.5% METHOCEL cellulose ethers at targeted doses of 10 and 500 mg/kg of body weight and volume of administration of 5 mL of dose solution/kg body weight. A measured amount of test substance was placed into a glass bottle and an appropriate volume of 0.5% METHOCEL added while stirring. A measure volume of 14C-Iabelled stock solution (in acetone) was then added, and the solution kept stirring at room temperature prior to capping to evaporate the acetone. The target radioactivity levels were approximately 500 µCi/kg body weight for the probe study and approximately 125 µCi/kg for the core metabolism study (25 µCi/rat). All 14C-labelled test substance dose solutions were analyzed for test substance by HPLC with UV detection, and radioactivity and homogeneity by liquid scintillation counting.

PREPARATION OF DOSING SOLUTIONS CONTAINING RADIOLABELLED TEST SUBSTANCE:
A non-radiolabelled test substance dose solution was prepared for the repeated dose segment of the core metabolism study. A measured amount of test susbtance was placed into a glass vial and an appropriate volume of 0.5% METHOCEL added while stirring. The dose solution was kept in a cold room (~20°C) with continuous stirring, and administered once daily for 14 consecutive days at a targeted dose of 10 mg/kg and volume of administration of 5 ml dose solution/kg of body weight. This solution was administered by gavage using a glass syringe and stainless steel feeding needle. Animals given repeated doses of nonradiolabelled test substance were weighed every 3-4 days and their dose volumes adjusted accordingly. The quantity of dose solution actually administered was determined volumetrically. The stability and concentration of test substance in the dose solution over the course of dosing was confirmed analytically.

Duration and frequency of treatment / exposure:

Groups 1 and 2 were given a single oral (gavage) dose of 10 and 500 mg/kg body weight of radiolabelled test substance, respectively. Group 3 was given 14 daily oral doses of 10 mg/kg non-radiolabelled test substance by gavage followed by a single 10 mg/kg oral (gavage) dose of radiolabelled test substance.

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

5 in core study, Two additional groups of 3 male rats each for bile samples collection study

Details on study design:

- Dose selection rationale: Dose levels were selected as outlined in the Pesticide Assessment Guideline-Subdivision F (EPA, 1984). The 10 and 500 mg/kg dose levels were selected since they span the dose range of toxicological interest. The low dose level of 10 mg/kg was the No-Observed-Effect-Level (NOEL) in a 13-week toxicity study in this strain of rat. The high dose level of 500 mg/kg exceeded the dose which produced treatment-related hepatic effects in a 13-week toxicity study.
- Rationale for animal assignment: Animals were randomly assigned to treatment groups using a computer-driven randomization procedure and were then individually identified by a uniquely numbered metal ear tag.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes, bile and Upon sacrifice, the core metabolism animals were exsanguinated via cardiac puncture, and the following tissues collected and analyzed for radioactivity: bone, brain, perirenal fat, gonads, heart, kidneys, liver, lung, blood, skeletal muscle, spleen, GI tract, GI tract contents, skin and the remaining carcass.
- Time and frequency of sampling: Blood: 0.25, 0.5, 0.75, 1.0, 1.5, 3, 6, 12, 24 and 48 hr post-dosing; Urine: 0-12 and 12-24 hr collection intervals; Feces: Probe study: 12 hr intervals through 48 hr and as a single specimen from 48-72 hr, core metabolism study: 24 hr intervals through 48 hr; bile excretion study: for a single 24 hr interval.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, tissues, cage washes, bile
- Time and frequency of sampling: Urine: 0-12 and 12-24 hr collection intervals
- From how many animals: Blood: Approximately 500 µL of blood (pooled 100 µL per rat), Urine samples were pooled (25-50 µL aliquots), Pooled fecal homogenates (1:3, feces:water), Pooled bile samples (25-45 µL aliquots)
- Method type(s) for identification: urine specimens were analyzed for radioactivity by liquid scintillation counting and subsequently analyzed by HPLC to determine the presence of metabolites. Identification of selected major metabolites was performed using HPLC and GC/MS. The composite fecal specimens were analyzed for 14C-activity by liquid scintillation counting and subsequently analyzed by HPLC to determine the presence of test substance and/or metabolites. Selected bile samples from each rat were analyzed for radioactivity, pooled by dose and time, and analyzed for parent test substance and metabolites by HPLC and GC/MS.

Statistics:

Only descriptive statistics will be conducted, i.e., Mean ± S.D. If appropriate, an elimination rate constant will be calculated from the interval 14C-excretion data by linear least squares analysis.

Preliminary studies:

A probe study was conducted using 2 groups of rats (1/sex/group) receiving 10 mg/kg of body weight (500 µCi/kgBW) of either 2-quinoline ring labeled or uniformally labeled phenyl ring,14C-test substance. The primary objective of the probe was to obtain excreta from treated rats to facilitate development of appropriate analytical techniques for the identification of test substance or metabolites. A secondary objective was to obtain preliminary information on the routes and rates of elimination of 14C-labelled test substance.
Results showed that no radioactivity was found in the expired organic traps for the 0-12 hr post-dosing period, and the total expired 14CO2 accounted for 0.55- 0.60% and 0.35-0.51% of the administered dose for the phenyl ring - and quinoline ring-labelled test substance, respectively. By 72 hr post-dosing, approximately 89% of the administered phenyl ring-labelled test substance and 84% of the quinoline ring-labelled test substance had been recovered. For the phenyl ring-labelled test substance, the urine accounted for 45-49% of the administered dose across gender, feces 38-40%, tissues and carcass ≤2% and final cage wash <2%. In contrast, the urine accounted for 15% of the administered quinoline ring-labelled test substance, the feces 66-68%, tissues
and carcass ≤2% and final cage wash ≤0.5%. Little difference was observed between male and female rats in the disposition of each radiolabelled form of 14C-labelled test substance. Based on the fact that for the 48-72 hr post-dosing interval ≤3% of the administered dose was recovered in the urine, feces, and as expired 14CO2, and only ~1.5% remained in the tissues and carcass at 72 hr, the core metabolism study duration was set at 48 hr. Overall, both forms of 14C-labelled test substance were rapidly absorbed and eliminated, with a greater percentage of the phenyl ring-labelled 1test substance eliminated in the urine due to the propensity of the fluorophenyl portion of the molecule for the urine

Type:

absorption

Results:

Approximately 85% and 60% of the 10 and 500 mg/kg doses were absorbed, respectively.

Type:

metabolism

Results:

Urine: Conjugates of 4-fluorophenol (4-FP) and 5,7-dichloro-4-hydroxyquinoline (DCHQ); Bile: Glucuronide and / or sulfate conjugates of two isomers of fluorophenyl ring-hydroxy-XDE-795

Type:

distribution

Results:

This rapid clearance is supported by the tissue distribution data which indicated that only ~2-9% of the dose remained in the body (GI tract contents, tissues and carcass) for all treatment groups upon sacrifice at 48 hr.

Type:

other: Elimination

Results:

The feces is major route of elimination as 68-78% of the dose was eliminated via this route in 48 hr. 13-20%was eliminated in the urine. The tissues and carcass accounted for 1-7%,GI tract contents <3% and final cage wash <1% of the administered dose.

Details on absorption:

Approximately 85% and 60% of the 10 and 500 mg/kg doses were absorbed, respectively. Comparable plasma concentrations of radioactivity were observed between the 10 mg/kg single and repeated male and female dose groups. Peak concentrations of radioactivity in the plasma were observed ~0.5-0.75 hr post-dosing for the 10 mg/kg groups. For the 500 mg/kg group, a more prolonged absorption phase was evident, with peak plasma concentrations of radioactivity observed at ~1.0-1.5 hr for male and female rats. Areas under the 14C-plasma concentration time curves (AUC's) for the 10 mg/kg single and repeated dose groups ranged from 22.3-30.4 µg eq.hr/g, and for the 500 mg/kg male and female rats 922 and 963 µg eq.hr/g, respectively

Details on distribution in tissues:

Comparison of the 10 mg/kg single and repeated dose groups showed a comparable distribution of tissue radioactivity. Fat contained the highest concentration of radioactivity, ranging from 0.1-0.3% of the administered dose/g of tissue, followed by the ovaries, liver, kidneys, GI tract and carcass, each containing ≤0.07% of the dose/g of tissue. The higher concentration of radioactivity in the ovaries may have been due to residual fat which may have remained with the tissue upon dissection. The concentrations of radioactivity for the 500 mg/kg dose group were slightly greater than proportional to the low dose in the fat (0.5-0.7% of the dose/g of tissue) GI tract, brain, carcass, skin, testes and ovaries, with concentrations generally proportional to the low dose found in the other tissues evaluated. Overall, any differences observed in the tissue concentrations of radioactivity between dose groups or gender were not thought to be of a magnitude to be biologically meaningful.The rapid clearance is supported by the tissue distribution data which indicated that only ~2-9% of the dose remained in the body (GI tract contents, tissues and carcass) for all treatment groups upon sacrifice at 48 hr.

Details on excretion:

Between 90-96% of the administered dose of quinoline ring-labelled test substance was recovered in the urine, feces, cage wash and tissues by 48 hr postdosing. The feces represented the major route of elimination as 68-78% of the dose was eliminated via this route in 48 hr, whereas 13-20% was eliminated in the urine. The tissues and carcass accounted for 1-7%,GI tract contents <3% and final cage wash <1% of the administered dose. By 24 hr post-dosing, 68-85% of the administered radioactivity had been recovered in the feces and urine for all treatment groups, supporting rapid excretion. A greater percentage of the dose was eliminated via the bile at 10 mg/kg (t1/2=4.5 hr) than at 500 mg/kg by 24 hr post-dosing. Correspondingly, a greater percentage of the dose was found as parent test substance in the feces of the high dose than low dose rats over this time interval.

Key result

Test no.:

#1

Toxicokinetic parameters:

other: Urinary half life

Remarks:

Urinary t1/2's ranged from 6-10 hr for all dose groups.

Key result

Test no.:

#2

Toxicokinetic parameters:

other: Plasma Half life

Remarks:

Plasma radioactivity t1/2's for the rapid and slow phases for the 10 mg/kg dose were <1 hr and 15-19hr, respectively, and 2-3 hr and 18-22hr for the 500 mg/kg dose

Key result

Test no.:

#3

Toxicokinetic parameters:

other: Bile Half life

Remarks:

A greater percentage of the dose was eliminated via the bile at 10 mg/kg (t1/2=4.5 hr) than at 500 mg/kg by 24 hr post-dosing.

Metabolites identified:

yes

Details on metabolites:

Test substance was extensively metabolized. Of the radioactivity in blood, ≤3% was found to be associated with parent test substance (AUC comparisons), indicating a high first pass metabolism. The major metabolites identified in this study were: urine, extensive cleavage of the diaryl-ether linkage of test substance resulting primarily in the formation of acid-labile conjugates of 4-fluorophenol (4-FP) and 5,7-dichloro-4-hydroxyquinoline (DCHQ), and lesser quantities of free DCHQ and 4-FP; bile, glucuronide and / or sulfate conjugates of two isomers of fluorophenyl ring-hydroxy-XDE-795; and feces, parent test substance and unconjugated forms of the same two isomers of fluorophenyl ring-hydroxy-XDE-795 as seen in the bile. No parent test substance was found in the urine and only a trace detected in the bile. Aside from evidence of limited absorption and saturation of the formation of 4-FP and observed biliary metabolites at 500 mg/kg, there appeared to be no substantive differences in the metabolism and disposition of test substance between gender or single and repeated exposure.

Following gavage administration of 10 mg 14C-labelled test substance, 54% of the administered dose was recovered in the bile 24 hr post-dosing, with an elimination t1/2 of 4.5 hr. Elimination via the feces represented 14% of the dose, urine 11%, followed by 5% in the carcass, 2% in the GI tract plus contents, 1% in the final cage rinse and <1% in the skin. In contrast at 500 mg/kg, only 21% of the dose was recovered in the bile 24 hr post-dosing. Due to the prolonged absorption phase observed at 500 mg/kg, a half-live for elimination in the bile was not estimated at this dose, but would be expected to be similar to that at 10 mg/kg once absorption was complete. Elimination via the feces represented 57% of the dose, urine ~3%, followed by 10% in the GI tract plus contents, 2% in the carcass and <1% in either the final cage wash or the skin 24 hr post-dosing. These results indicate that biliary excretion into the feces is an important route of elimination of test substance. Furthermore, they indicate lower absorption at the 500 mg/kg dose relative to that at 10 mg/kg.

Conclusions:

Absorption: Approximately 85% and 60% of the 10 and 500 mg/kg doses were absorbed, respectively
Distribution: Only ~2-9% of the dose is remained in the body (GI tract contents, tissues and caeacss) for all treatment groups upon scarefice at 48 hr.
Metabolism: Test substance was extensively metabolised. Of the radioactivity in blood, ≤3% was found to be associated with parent test susbatnce(AUC comparisons), indicating a high first pass metabolism.
Excretion: Between 90-96% of the administered dose of quinoline ring-labelled test substance was recovered in the urine, feces, cage wash and tissues by 48 hr post dosing. The feces represented the major route of elimination as 68-78% of the dose was eliminated via this route in 48 hr, whereas 13-20% was eliminated in the urine.

Executive summary:

The study was conducted according to OECD guideline 417 to assess pharmacokinetics and metabolism of radiolabeled test substance in the Fischer 344 rat. Groups of 5 rats/sex were given a single 10 or 500 mg/kg oral dose of quinoline ring-labelled test substance, or 14 daily doses of 10 mg/kg non-radiolabelled test substance followed by a single 10 mg/kg oral dose of 14C labelled test substance. Rats were sacrificed 48 hr after receiving the radiolabelled dose. An additional group of 3 male bile duct cannulated rats received a single dose of 10 or 500 mg/kg quinoline ring-labelled test substance and were sacrificed 24 hr later. One male and female rat/ group received 10 mg/kg of either phenyl ring- or quinoline ring-labelled test substance to assist in metabolite analysis. Between 90-96% of the administered dose of quinoline ring-labelled test substance was recovered in the urine, feces, cage wash and tissues by 48 hr post dosing. The feces represented the major route of elimination as 68-78% of the dose was eliminated via this route in 48 hr, whereas 13-20% was eliminated in the urine. The tissues and carcass accounted for 1-7%, GI tract contents <3% and final cage wash <1% of the administered dose. Urinary t1/2's ranged from 6-10 hr for all dose groups. Plasma radioactivity t1/2's for the rapid and slow phases for the 10 mg/kg dose were <1 hr and 15-19 hr, respectively, and 2-3 hr and 18-22 hr for the 500 mg/kg dose. By 24 hr post-dosing, 68-85% of the administered radioactivity had been recovered in the feces and urine for all treatment groups, supporting rapid excretion. A greater percentage of the dose was eliminated via the bile at 10 mg/kg (t1/2=4.5 hr) than at 500 mg/kg by 24 hr post-dosing. Correspondingly, a greater percentage of the dose was found as parent test substance in the feces of the high dose than low dose rats over this time interval. It was estimated that approximately 85% and 60% of the 10 and 500 mg/kg doses were absorbed, respectively. Test substance was extensively metabolized. Of the radioactivity in blood, ≤3% was found to be associated with parent test substance (AUC comparisons), indicating a high first pass metabolism. The major metabolites identified in this study were: urine, extensive cleavage of the diaryl-ether linkage of test substance resulting primarily in the formation of acid-labile conjugates of 4 fluorophenol (4-FP) and 5,7- dichloro-4-hydroxyquinoline (DCHQ), and lesser quantities of free DCHQ and 4-FP; bile, glucuronide and / or sulfate conjugates of two isomers of fluorophenyl ring-hydroxy-XDE-795; and feces, parent test substance and unconjugated forms of the same two isomers of fluorophenyl ring-hydroxy-XDE-795 as seen in the bile. No parent test substance was found in the urine and only a trace detected in the bile. Aside from evidence of limited absorption and saturation of the formation of 4-FP and observed biliary metabolites at 500 mg/kg, there appeared to be no substantive differences in the metabolism and disposition of test substance between gender or single and repeated exposure.

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 (500 mg/kg bw) dose administration and after multiple treatments at low doses. Between 90-96% of the administered dose was recovered in the urine, feces, cage wash and tissues by 48 hr post dosing. The feces represented the major route of elimination as 68-78% of the dose was eliminated via this route in 48 hr, whereas 13-20% was eliminated in the urine. The tissues and carcass accounted for 1-7%, GI tract contents <3% and final cage wash <1% of the administered dose. It was estimated that approximately 85% and 60% of the 10 and 500 mg/kg doses were absorbed, respectively. Test substance was extensively metabolized. Of the radioactivity in blood, ≤3% was found to be associated with parent test substance, indicating a high first pass metabolism. The major metabolites identified in this study were: urine, extensive cleavage of the diaryl-ether linkage of test substance resulting primarily in the formation of acid-labile conjugates of 4 fluorophenol (4-FP) and 5,7- dichloro-4-hydroxyquinoline (DCHQ), and lesser quantities of free DCHQ and 4-FP; bile, glucuronide and / or sulfate conjugates of two isomers of fluorophenyl ring-hydroxy-XDE-795; and feces, parent test substance and unconjugated forms of the same two isomers of fluorophenyl ring-hydroxy-XDE-795 as seen in the bile. No parent test substance was found in the urine and only a trace detected in the bile. Aside from evidence of limited absorption and saturation of the formation of 4-FP and observed biliary metabolites at 500 mg/kg, there appeared to be no substantive differences in the metabolism and disposition of test substance between gender or single and repeated exposure.