Isopropylcyclohexane

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

other: supporting study about metabolism of isopropylcyclohexane

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.

Data source

Reference

Reference Type:

publication

Title:

Metabolism of nephrotoxic isopropylcyclohexane in male Fischer 344 rats

Author:

Henningsen GM, Salomon RA, Yu KO, Lopez I, Roberts J and Servé MP

Year:

1988

Bibliographic source:

J. Toxicol. Environ. Health 24, 19-25

Materials and methods

Principles of method if other than guideline:

Animals, Housing, and Exposure Conditions
Eighteen Fischer 344 male rats weighing 310 ± 10 g were divided into 2 groups of 12 treated rats and 6 control rats. Rats were maintained in individual cages in a controlled environment with temperatures of 74 ± 2°F, relative humidity of 55 ± 10%, and a light cycle of 12 h on and 12 h off. When urine was not being collected in metabolism cages, rats were housed three to a polycarbonate cage with hardwood chips for bedding. Feed and water were available ad libitum. The highest tolerated dose of isopropylcyclohexane was determined to be 900 mg/kg in preliminary acute range-finding studies. Hence near-maximum tolerated doses of 800 mg/kg of neat isopropylcyclohexane were administered by oral gavage to 12 rats on alternate days for 14 d to facilitate
metabolite identification and to induce rapidly any potential renal toxic effects. Six controls animals were given equivalent volumes of water over the same duration. Following the 14-d exposure period, the rats were sacrificed by a saturated halothane overdose and the kidneys were excited 24 h following the final dose. Histopathologic examination was performed on paraffin-embedded kidney sections stained with hematoxylin and eosin. Tissues from treated rats were compared to controls for characteristic lesions of hydrocarbon-induced nephropathy, including hyaline droplet formation, tubular cysts, and papillary calcification. Lesions were graded by pathologists for degree of severity. Isolation of Urinary Metabolites For the first 48 h following initial dosing, rats were placed in metabolism cages where the excreted urine was collected and then frozen until analyzed. The pH of urine samples was adjusted to 4.0, and 0.5 ml glucuronidase/sulfatase (specific activity of 5.5 and 1.5 units/ml, respectively; Calbiochem, Lajolla, Calif.) was added to the 5.0-ml aliquots. This
amount of enzyme was in excess of that needed for complete hydrolysis of conjugates, as determined by varying the quantities added to different aliquots of samples. The solution was shaken at 37°C for 16 h, cooled at room temperature, and filtered through a diatomaceous earth column (Clin Elut, Analytichem International, Harbor City, Calif.) using neat methylene chloride as the eluent. Unconjugated metabolites of isopropylcyclohexane were isolated by direct extraction of a second 5.0- ml aliquot of urine with methylene chloride. The urine extract samples were then analyzed by gas chromatography and gas chromatograph/mass spectometry. Urinary Metabolite Identification The methylene chloride extracts of urine metabolites were analyzed on a gas chromatograph equipped with a flame ionization detector (model 5880A, Hewlett-Packard Corp., Avondale, Pa.) and using 25|x|0.22 m ID carbowax 20 M fused silica capillary column (Hewlett-Packard Corp., Avondale, Pa.). A 60°C oven temperature was maintained for 1 min after sample injection and then programmed at
2°C/min to 180°C. Detector and injection port temperatures were 250°C and 200%C, respectively. Helium was used as the carrier gas, with a linear velocity of 22 cm/s at 100%C and a split ratio of 5:1. Metabolite identification was conducted with a Hewlett-Packard 5985 gas chromatograph/mass spectrometer system and a 4 ft x 2 mm ID 3% SP-1000 and 100/120 Supelco-port glass column (Supelco Corp., Bellefonte, Pa.) with a helium flow rate of 28 ml/min. The oven temperature was held at 100%C for 1 min and then programmed at 10°C/min at 200°C. The injection-port temperature was 200°C. The mass spectrometer was a quadrupole instrument operated in the electron impact mode at a voltage of 70 eV and with an ion source temperature of 200°C.
Identification of both treated and control sets of urinary metabolites was confirmed by comparing mass spectra fragmentation patterns with the fragmentation patterns of purchased or synthesized compounds. Quantitation of the metabolites was accomplished by taking weighed samples of the individual metabolites, adding a weighed amount of tridecane, and determining the relative gas chromatograph areas. A weighed sample of tridecane was then added to the urine samples before analysis, and the ratio of the gas chromatograph areas was compared to yield the relative molar abundancies.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isopropylcyclohexane

EC Number:

211-792-4

EC Name:

Isopropylcyclohexane

Cas Number:

696-29-7

Molecular formula:

C9H18

IUPAC Name:

(propan-2-yl)cyclohexane

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

Animals, Housing, and Exposure Conditions
Eighteen Fischer 344 male rats weighing 310 ± 10 g were divided into 2 groups of 12 treated rats and 6 control rats. Rats were maintained in individual cages in a controlled environment with temperatures of 74 ± 2°F, relative humidity of 55 ± 10%, and a light cycle of 12 h on and 12 h off. When urine was not being collected in metabolism cages, rats were housed three to a polycarbonate cage with hardwood chips for bedding. Feed and water were available ad libitum. The highest tolerated dose of isopropylcyclohexane was determined to be 900 mg/kg in preliminary acute range-finding studies. Hence near-maximum tolerated doses of 800 mg/kg of neat isopropylcyclohexane were administered by oral gavage to 12 rats on alternate days for 14 d to facilitate metabolite identification and to induce rapidly any potential renal toxic effects.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

The highest tolerated dose of isopropylcyclohexane was determined to be 900 mg/kg in preliminary acute range-finding studies. Hence near-maximum tolerated doses of 800 mg/kg of neat isopropylcyclohexane were administered by oral gavage to 12 rats on alternate days for 14 d to facilitate
metabolite identification and to induce rapidly any potential renal toxic effects. Six controls animals were given equivalent volumes of water over the same duration.

Duration and frequency of treatment / exposure:

14 days, with oral gavage on alternate days

Doses / concentrations

Remarks:

Doses / Concentrations:
800 mg/kg of neat isopropylcyclohexane

No. of animals per sex per dose / concentration:

12 treated rats

Control animals:

yes

Results and discussion

Main ADME results

Type:

metabolism

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Metabolites
Fischer 344 male rats were exposed subacutely to isopropylcyclohexane and produced the urinary metabolites. The
isopropylcyclohexane molecule was metabolically oxidized on both the ring and the isopropyl side chain, with oxidative attack on the cyclohexane ring predominating by a factor of two (42.4:21.3 mol). Metabolic oxidation of the cyclohexane ring produced both cis- and trans-4-isopropylcyclohexanol, with the trans isomer being the major overall metabolite. There was no trace of any isomeric form of 20 or 3-isopropylcyclohexanol. The cyclohexanediols that were isolated as urinary metabolites showed a high degree of stereochemical specificity.

Urinary Metabolites of Fischer 344 Male Rats Dosed with Isopropylcyclohexane
Metabolite

cis-4-lsopropylcyclohexanol
trans-4-lsopropylcyclohexanol
2-Cyclohexylpropanoic acid
2-Cyclohexyl-1,3-propanediol
2c-Hydroxy-4c-isopropylcyclohexanol
2c-Hydroxy-4t-isopropylcyclohexanol
2t-Hydroxy-4t-isopropylcyclohexanol
ane ring predominating by a factor of two (42.4:21.3 mol). Metabolic oxidation of the cyclohexane ring produced both cis- and trans-4-
isopropylcyclohexanol, with the trans isomer being the major overall metabolite. There was no trace of any isomeric form of 20 or 3-isopropylcyclohexanol. The cyclohexanediols that were isolated as urinary metabolites showed a high degree of stereochemical specificity.

Any other information on results incl. tables

Histology Histopathologic findings for male Fischer 344 rats dosed with isopropylcyclohexane consisted of the following: (a) a moderate but variable accumulation of hyaline droplets in the cytoplasm of tubular epithelial cells; (b) individual tubular epithelial degeneration; and (c) tubular cyst formation at the corticomedullary junction. Minimal sporadic changes, excluding cysts, were found in the water-dosed control rats.