5-Isothiazolecarboxamide, 3,4-dichloro-N-(2-cyanophenyl)-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jul 2004 - 8 Jun 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japan Ministry of Agriculture Forestry and Fisheries (JMAFF) guideline (12, Noshan number 8147)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dichloro-N-(2-cyanophenyl)-1,2-thiazole-5-carboxamide

EC Number:

619-682-1

Cas Number:

224049-04-1

Molecular formula:

C11H5Cl2N3OS

IUPAC Name:

3,4-dichloro-N-(2-cyanophenyl)-1,2-thiazole-5-carboxamide

Constituent 2

Reference substance name:

3,4-dichloro-2'-cyano-1,2-thiazole-5-carboxanilide

IUPAC Name:

3,4-dichloro-2'-cyano-1,2-thiazole-5-carboxanilide

Radiolabelling:

yes

Remarks:

Two different labelled forms: [Phenyl(U)-14C]-test substance with 14C on the phenyl ring and [lsothiazole-3-14C,Carboxamide-14C]-test substance with 14C on the 3 position of the isothiazole ring and the carboxamide

Test animals

Species:

rat

Strain:

other: Crl:Wl® (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, lnc., Raleigh, NC
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 198-363 g
- Housing: Individually metabolism cages
- Individual metabolism cages: Yes
- Diet (e.g. ad libitum): Lab Diet Feeds certified rodent diet No. 5002 ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 2% cremophor EL in HPLC grade water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): A solution of Cremophor EL 2% (v/v) in HPLC grade water was used as the vehicle for the test material since it provides homogeneous doses and is a standard vehicle used in toxicology studies.
- Amount of vehicle (if gavage): 10 mL/kg

Duration and frequency of treatment / exposure:

Single dose, termination time up to and including 168 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
4 and 200 mg/kg bw

No. of animals per sex per dose / concentration:

Pilot Pharmacokinetic Experiment: 1
Pilot Excretion Experiment: 2
Pharmacokinetic Experiment: 4
Single Dose Excretion and Distribution Experiments (maximum termination times): 4
Tissue Distribution Time Course Experiment (intermediate termination times): 3

Control animals:

yes, concurrent vehicle

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Expired air, urine, faeces, blood, plasma, serum or other tissues, cage washes, bile.
- Time and frequency of sampling: 6, 12 and 24h and in 24h interval until termination, blood collection at 0.25, 0.5, 1, 2, 6, 12, 24, 48, 72 and 120 h after dosing and at all termination time points.
- other: At termination, the following tissues were excised from the animals used in the Pilot Excretion Experiment, the Single Dose Excretion and Distribution Experiments and the Tissue Distribution Time-Course Experiment: adrenal glands, bone (femur), bone marrow (femur), brain, fat, heart, stomach, intestine, kidneys, liver, lungs, muscle, ovaries, pancreas, pituitary gland, prostate gland, spleen, testes, thymus, thyroid, uterus, skin with hair, mandibular gland, eye balls, spinal cord, sciatic nerve, whole blood, blood cells, and plasma.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine, faeces, cage wash.
- Time and frequency of sampling: in 24h intervals.
- From how many animals: (samples pooled or not): Pooled.
- Sample preparation: Pooled excreta samples from selected time points were chosen for extraction and chromatographic analysis:
Urine: Hydrolysis with sulfatase enzyme (Type H-l from Helix pomatia) that has both sulfatase and glucuronidase activities;
Cage wash: Aliquots of the pooled cage wash samples from the lsothiazole label were concentrated under nitrogen and then analyzed by HPLC Method using a radiochemical detector equipped with a solid cell. Aliquots from the pooled cage wash samples containing the Phenyl label were analyzed (without concentration) by HPLC Method using a radiochemical detector equipped with a liquid cell.
Faeces: The pooled samples were extracted two times with acetonitrile, three times with 50% acetonitrile/warter containing 1% formic acid and one time with 50% methanol/water containing 1% formic acid. The ratio of the sample to solvent was 1:3 (wt:vol). After suspension in solvent, the samples were sonicated for 10 minutes, vortexed for l minute and then centrifuged at ~3000-5000 rpm for 10 minutes. The supernatant was transferred to a graduated cylinder, the volume was recorded and duplicate aliquots were taken for LSC. The supernatants from each extraction step were combined and concentrated by rotary evaporation. PES was analyzed by combustion for quantitation of unextractable residues.
Liver: Pooled liver samples were subjected to solvent extraction using the same method as used for the faeces samples. The extracts were taken to dryness by rotary evaporation and reconstituted in appropriate combination of acetonitrile/water. They were centrifuged and an aliquot was taken for LSC. An aliquot of the supernatant was taken for HPLC. If the total amount of radioactivity in the samples was low, chromatographic fractions of one-minute each were collected and analyzed by LSC to obtain radiohistograms. The postextraction solids (PES) were analyzed for radioactivity by LSC following combustion analysis of aliquots.
Kidney: Pooled kidney samples were subjected to solvent extraction rising the same method as used for the faeces samples. The extracts were taken to dryness by rotary evaporation and reconstituted in appropriate combination of acetonitrile/water. They were centrifuged and an aliquot was taken for LSC. Each supernatant was analyzed by HPLC. Total radioactivity in post»extraction solids (PES) quantitated combustion analysis followed by LSC.
Plasma: The pooled blood samples were separated into plasma and packed cells by centrifugation.
Aliquots (2 x l0 µL) of the pooled plasma samples were analyzed by LSC. Plasma samples from the 0.25 and 12-hour time points for the 4 mg/kg dose-group and the 0.5 and 24-hour 200 mg/kg dose dose-group (except for the samples with low levels of radioactivity) were analyzed by HPLC. The pooled plasma samples were prepared for analysis by solid phase extraction using Varian BondElut C18, 500 mg 3 mL columns (Varian Inc., Harbor City, CA).

- Method type(s) for identification: Liquid scintillation counting, Combustion analysis, HPLC-RAD, HPLC-LSC, LC-MS-MS, NMR.

Statistics:

Pharmacokinetic: WinNonlin calculation

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

30-36% (4 mg/kg bw), 10-14% (200 mg/kg bw) after 168 hours (calculated from the % of the dose recovered in urine, cage wash and tissues)

Main ADME results 2

Type:

excretion

Results:

91 - 96% after 48 hours

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Total recovery for the isothiazole label was around 93% for the 4 mg/kg bw group and 98% for the 200 mg/kg bw group. For the phenyl label >92% for the 4 mg/kg bw and around 95% for 200 mg/kg bw group had been recovered.
The maximum plasma concentration (Cmax) of 0.2 - 0.36 µg test substance equivalents/g administered dose following the 4 mg/kg treatment was reached at 0.3 to 1.7 hours. Following the 200 mg/kg treatment the highest plasma concentration of 2.57 - 4.17 µg test substance equivalents/g administered dose was reached at 0.9-10.6 hours. Elimination half-lives (tm) were marginally longer at the 200 mg/kg close level (17.9-20.5 hours) than at the 4 mg/kg dose level (13.9-17.8 hours). There were slight differences in the genders at both dose levels. For the female rats values were greater than the corresponding values for male rats. The relative systemic exposure for the two dose levels, as determined by comparison of plasma Cmax and AUC values, was dose-dependent (nonlinear).
For the female rats values were greater than the corresponding values for male rats. The relative systemic exposure for the two dose levels, as determined by comparison of plasma Cmax and AUC values, was dose-dependent (nonlinear).

Details on distribution in tissues:

After 168-h exposure period, total residues in tissues were below 0.2 % of the administered dose and total residues in the residual carcass were 0.12% of the administered dose or less. Within the 4 mg/kg dose group, the highest residue levels were found in liver (0.134 ppm) and kidney (0.068 ppm). Residue levels for all other tissues were less than 0.03 ppm or were not detectable for the 4 mg/kg dose level. No sex-related differences in tissue residue levels were observed.
Within the 200 mg/kg dose group, the highest residue levels in male rats were found in stomach (8.95 ppm), intestine (1.29 ppm), kidney (1.29 ppm), liver (4.92 ppm) and packed cells (0.98 ppm). Residue levels for all other tissues were less than 0.6 ppm or not detectable for the 200 mg/kg dose level. No sex-related differences in tissue residue levels were observed.

Details on excretion:

Isothiazole label:
4 mg/kg: male: 58% after 24 hours, 90 % after 48 hours and 93% after 168 hours, female: 79% after 24 hours, 91% after 48 hours and 93% after 168 hours.
200 mg/kg: male: 60% after 24 hours, 97% after 48 hours and 99% after 168 hours; female 59% after 24 hours, 96% after 48 hours and 98% after 168 hours.
Phenyl label:
4 mg/kg: male: 64% after 24 hours, 86 % after 48 hours and 93% after 168 hours, female: 82% after 24 hours, 104% after 48 hours and 106% after 168 hours.
200 mg/kg: male: 47% after 24 hours, 90% after 48 hours and 93% after 168 hours; female 65% after 24 hours, 95% after 48 hours and 97% after 168 hours.

Toxicokinetic parameters

Toxicokinetic parameters 1

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 4 mg/kg bw: 15.8 - 25.1 h

Toxicokinetic parameters 2

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 200 mg/kg bw: 13.6 - 40.2 h

Toxicokinetic parameters 3

Test no.:

#1

Toxicokinetic parameters:

AUC: 4 mg/kg bw: 3.87 - 4.84 µg x h/g

Toxicokinetic parameters 4

Test no.:

#2

Toxicokinetic parameters:

AUC: 200 mg/kg bw: 65.19 - 111.82 µg x h/g

Toxicokinetic parameters 5

Test no.:

#1

Toxicokinetic parameters:

Cmax: 4 mg/kg bw: 0.2 - 0.36 µg/g

Toxicokinetic parameters 6

Test no.:

#2

Toxicokinetic parameters:

Cmax: 200 mg/kg bw: 2.57 - 4.17 µg/g

Toxicokinetic parameters 7

Test no.:

#1

Toxicokinetic parameters:

Tmax: 4 mg/kg bw: 0.3 - 1.7 h

Toxicokinetic parameters 8

Test no.:

#2

Toxicokinetic parameters:

Tmax: 200 mg/kg bw: 0.9 - 10.6 h

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Isothiazole label:
Urine: 3,4-dichloro-5-isothiazolecarboxylic acid; glucuronide of 4’-OH- and 4’-OH-metabolites of test substance, respectively.
Faeces: Unmetabolised test substance (main 14C component), and to a minor degree 4’-OH- and 4’,5’-OH-metabolites of test substance, respectively.
Phenyl label:
Urine: 2-amino-5-hydroxybenzonitril; glucuronide of 4’-OH-, 4’-OH- and 4’,5’-OH-metabolites of test substance, respectively.
Faeces: Unmetabolised test substance (main 14C component), and to a minor degree 4’-OH- and 4’,5’-OH-metabolites of test substance, respectively.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results