4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 February 1995 - 15 August 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA Pesticide Assessment Giudelines, Subdivision F, 40 CFR Part 158, 85-1

Version / remarks:

October 1982

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

EC Number:

605-140-1

Cas Number:

158237-07-1

Molecular formula:

C16H20ClN5O2

IUPAC Name:

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Remarks:

BOR:WISW

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann Versuchstierzucht GmbH & Co KG, Borchen, Germany
- Age at study initiation: approximately 8 weeks (males), approximately 11 weeks (females)
- Weight at study initiation: about 200 g
- Housing: 5 animals in Makrolon type III cages (acclimatization period), individually in Makrolon metabolism cages (during study)
- Diet: Altromin 1324 standard feed for rats, 18 g per animal/day
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 -25
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: acetonitile and 0.5% aqueous tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The radiolabelled compound was dissolved in acetonitrile and stored at approximately 4°C. The non labelled test compound was stored in solid form. For preparation of each dose the labelled test compound in acetonitrile was pipetted and blown dry with a gentle stream of nitrogen prior to adding 0.5% aqueous tragacanth suspension. Preparation of a homogenous suspension required agitation of the test substance with the tragacanth suspension at 50°C to 70°C for at least 15 min using supersonics and/or stirring at the same temperature for approximately 16 h. If needed non-labelled parent compound was weighed, dissolved in acetonitrile and mixed with the radioactive test solution prior to evaporation with nitrogen.

Duration and frequency of treatment / exposure:

single administration

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

males and females

Doses / concentrations 2

Dose / conc.:

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

Remarks:

males

No. of animals per sex per dose / concentration:

5 (low dose, both males and females)
5 (high dose, males)

Control animals:

no

Details on study design:

- Dose selection rationale: considering the low toxicity and the limited solubility of the compound the low dose was set at 1.5 mg/kg bw/day and the high at 75 mg/kg bw/day

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: expired air, urine, cage washes, faeces, blood, plasma, serum, organs and tissue (erythrocytes, spleen, gastrointestinal tract, bone, lung and muscle, adrenal glands, thyroid, ovaries, renal fat and uterus).
- Time and frequency of sampling: 0-4, 4-8, 8-24, 24-48 and 48-72 h (for tests 1 and 2) postadministration (urine), 0-24, 24-48 and for tests 1 and 2 also 48-72 h after dosing (faeces), collected 0.08, 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 24, 32, 48 h and for test 1 also 72 h after administration (blood), 0-8, 8-24, 24-48 and 48-72 h after dosage test 1(expired air), at necropsy 72 h postadministration (test 1,2) and 48 h postadministration (test 3,4), organs and tissues were homogenized for analysis.
- Other: in tests 1 and 2 animals were sacrificed 72 h postadministration, test 3 and 4 were shortened to 48 h thereafter, because of a rapid excretion of radioactivity in the first tests.
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, cage washes, faeces.
- Time and frequency of sampling: 0-48 h collection period and 0-24 h collection period (urine), 0-24 h collection period (faeces).
- From how many animals: 0-48 h collection period, urine of rats of test 1 anf 3 were combined for analysis of unknown metabolites, aliquots of urine of the collection periods 0 - 48 h of the rats of tests no. 1, 2, 3, and 4 (0 -24 h) were combined to separate samples per dose group and sex (urine), faeces of cellection period 0 - 24 h were combined per dose group and sex of tests 1 to 4 (faeces).
- Method type(s) for identification: HPLC-MS-MS, TLC, NMR-Spectroscopy.
- Limits of detection and quantification: a radioactive peak was regarded as relevant having a signal to noise ratio of at least 2.5.

Results and discussion

Preliminary studies:

In a preliminary test the expiration of CO2 and other labelled volatiles was followed over a period of 72 h using male rats (test 1). In this test < 0.05% of the administered dose were expired within 72 h.

Main ADME results

Main ADME results 1

Type:

absorption

Results:

The test substance was absorbed rapidly from the gastrointestinal tract of rats after oral administration. In the high dose group absorption was at least 44% in males and >80% in low dose male and female rats.

Main ADME results 2

Type:

distribution

Results:

< 5% of the administered dose remained in the body in males and 7% and more in females, liver followed by kidneys, lungs and thyroid gland contained significant amounts.

Main ADME results 3

Type:

metabolism

Results:

The test substance was metabolized extensively. Two major groups of metabolites (the hydrolysis product containing the radiolabelled moiety either as free Cyclohexyl ethylamine (CEA) and Cyclohexylamine (CA) or as hydroxylated CEA) were identified.

Main ADME results 4

Type:

excretion

Results:

Total excretion was rapid. >92% was excreted within 48 h or 72 h at both doses in urine and faeces. The ratio between renal to faecal excretion was 4:1 to 3:1 in males and females.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Absorption of the radio-labelled test substance started immediately after dosing and was absorbed rapidly from the gastrointestinal tract of the rats in all dose groups after oral administration of a suspension in 0.5% aqueous tragacanth. The absorption of the test substance amounted to at least 44% (high dose male animals) and >80% (low dose male and female animals), given as the renal portion plus the sum of the organs without gastro-intestinal tract. The maximum equivalent concentration was reached about 70 to 100 min postadministration in the low dose groups and at 180 min in the high dose males. The dose normalized equivalent concentration peaked at P=0.14 for the high dose group and at P=0.78 and 0.54 for the low dose groups and thus between 14% and 78% of the equidistribution concentration when comparing the average values for five animals in each test. The plasma maximum was independent of the sex and not proportional to the administered dose, the latter indicating incomplete absorption and unchanged parent test compound in the faeces of the high dose animals.

Details on distribution in tissues:

At both doses a moderately fast decrease of redioactivity concentration in plasma, due to fast distribution and excretion was observed. Elimination half-lives were relatively short and ranged from about 16 to 37 h. The total clearance was relatively high in all dose groups, 1.1 to 7.5 [mL/(min*kg bw)] without correction for the bioavailability. The renal clearance (CLR = CL x % renal excretion) was moderately high, 0.7 and 7.5 [mL/(min*kg bw)]. These numbers are in the range of the maximum glomerular filtration capacity of the kidney meaning that the bulk of radioactivity was eliminated via glomerular filtration without reabsorption. It was shown that the different dose levels seem to influence the distribution behaviour of total radioactivity in the body. The mean residence time MRT of the total radioactive dose in the plasma was short in all dose groups, about 24 and 43 hours, indicating that elimination is a moderately fast process. The radioactivity remaining in the body without the gastrointestinal tract was 4.5% to 4.0% of the dose at sacrifice in low dosed males (tests 1 and 2), 6.7% for low dosed females (test 3) and 2.8% for high dosed males. Significant amounts of radioactive residues were found in the liver and to a minor extent in the kidney. The residue in males was 0.7 µg/g and in females 1.2 µg/g (low dose group) and 7.2 µg/g in high dosed males. Among the organs and tissues checked liver in the low dose group was found to contain the highest dose normalized equivalent concentration of P = 0.45 for the males and P = 0.89 for the females, i.e. 45% and 89% of the equidistribution concentration whereas it amounted to 0.17 for the (high dose males, thus indicating a much lower absorption at the high dose level. Kidney exhibited between 21% and 32% of the radioactivity in the liver. In the low dose groups also the lungs and the thyroid gland showed relatively high residues.

Details on excretion:

After oral administration on average 92.7% of the recovered radioactivity was excreted via urine and faeces within 48 h of the female animals and 95.4% or more within 72 h of the low dose males and 97.0% of the high dose males after 48 h. The majour route of excretion was renal (ratio renal to faecal in in low dose groups: 3.9:1 to 5.5:1 and in high dose groups 0.7:1). The excretion pattern of total radioactivity was not significantly different between male and female rats. Only slightly different retention behaviour of radioactive residues was observed between male and female rats: females retained almost twice the amount as compared to males.

Toxicokinetic parameters

Toxicokinetic parameters 1

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 16.1 h

Toxicokinetic parameters 2

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 37.0 h

Toxicokinetic parameters 3

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 16.4 h

Toxicokinetic parameters 4

Test no.:

#2

Toxicokinetic parameters:

AUC: 24.6

Remarks:

µg/(gxh)

Toxicokinetic parameters 5

Test no.:

#3

Toxicokinetic parameters:

AUC: 25.9

Remarks:

µg/(gxh)

Toxicokinetic parameters 6

Test no.:

#4

Toxicokinetic parameters:

AUC: 166

Remarks:

µg/(gxh)

Toxicokinetic parameters 7

Test no.:

#2

Toxicokinetic parameters:

Cmax: 1.01

Remarks:

µg/g

Toxicokinetic parameters 8

Test no.:

#3

Toxicokinetic parameters:

Cmax: 1.71

Remarks:

µg/g

Toxicokinetic parameters 9

Test no.:

#4

Toxicokinetic parameters:

Cmax: 6.38

Remarks:

µg/g

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

In the low dose group unchanged test substance was only detected at minor acounts in faeces. In high dosed males unchanged test substance was observed as major component in faeces extracts. In the urine no parent compound was detectable. Main metabolites identified in urine were cyclohexyl-ethylamine (CEA) and cyclohexylamine (CA). These free amines accounted for 7.5% and 4.2% (CEA, CA, test 4), 18.3% and 13.4% (test 3) and 9.7% to 12.4% and 10.7 to 11.7% (test 2, test 1) in the sum of urine and faeces extracts, females showing a slightly different pattern with less hydroxylated and more unchanged CEA and CA. In addition six minor metabolites (0.2% to 4.1%) were identified in urine. They belong to two groups, one baring the CEA-carbamoyl moiety linked to amino groups of endogenous acids (cyclohexyl ethyl carbamoyl-tauric acid [CEA-CO-tauric], cyclohexyl ethyl carbamoyl glutamic acid [CEA-CO-glutamic] , hydroxy-cyclorjexyl ethyl carbamoyl glutamic acid [HO-CEA-COglutamic] and cyclohexyl ethyl carbamoyl hydroxyglutamic acid [CEA-CO-HO-glutamic]) and the other one baring the CA-carbamoyl group linked to the nitrogen of ammonia (cyclohexylurea), urea (cyclohexylbiuret) and the sulfur of mercapturic acid (cyclohexyl carbamoyl mercapturic acid [CA-CO-S-cys-N-ac]). No sulfate nor glucuronic acid conjugates were found. In males of the high dose group a proportionally lower amount of metabolites were found in faeces, but apprimately 19% of unchanges parent compound was detected. The total rate of identification was relatively high in the low dose group (68% to 78%) of the administered dose (73% to 75% of the total recovered). The identification was lower in the high dose group (63% of the dose and 64% of the recovered). In the low dose tests the sum of the main metabolites hydroxy-cyclohexyl-ethylamine (HO-CEA), CEA and CA amounted to 62% to 65% of the recovered radioactivity, females showing more of the unhydroxylated amines than males. In the high dose test the unchanged substance was a major compound and was about 61% together with the three amines. The other metabolites found preferentially in urine were minor to trace metabolites with lower relative amounts in the high dose test.