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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, minor restrictions in design and/or reporting but otherwise adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Objective of study:
other: Absorption, distribution, metabolism, excretion and tissue retention
Principles of method if other than guideline:
Route/rate of excretion following oral/i.v. radiolabelled substance
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
14C-cyclohexane – supplied by NIEHS, prepared by Midwest Research Institute as a 10% solution in 95% ethanol and at a specific activity of 5.3mCi/mmole. Unlabelled cyclohexane was Fisher reagent grade.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Adult rats, weights recorded at the time of dosing. Diet- certified Purina Rat Chow, water - ad libitum. The rats were examined for signs of disease or abnormality upon arrival and quarantined at least two weeks before they were used in a study. Rats were housed in individual glass metabolism chambers from the day before they were used in an experiment.

Administration / exposure

Route of administration:
other: Oral: gavage, Intravenous: tail vein
Vehicle:
other: Oral: sesame oil, Intravenous: serum from male Fisher 344 rats
Duration and frequency of treatment / exposure:
Single dose
Doses / concentrations
Remarks:
Doses / Concentrations:
Oral: 100, 200, 1000 or 2000mg per kg bodyweight (14C)
Intravenous: 9.7-11.1mg kg bodyweight (14C)
No. of animals per sex per dose:
Oral: 2 rats at 100, 25 at 200, 3 at 1000 and 3 at 200mg of [14C]-cyclohexane per kg bodyweight
Intravenous: 3 rats at 10mg of [14C]-cyclohexane per kg bodyweight
Control animals:
no
Details on dosing and sampling:
The concentration of [14C]-cyclohexane in the dosing solution was determined immediately after each dose was administered. These individual values were used to calculate the dose for the particular animal which had just been dosed.

Volatile organics in breath were collected by pulling air through the metabolism cage at 200-500mL/min and then through a series of 3 traps; the traps were changed at intervals up to 72 hours.

Urine was collected in round-bottom flasks maintained in ice baths at intervals up to 72 hours.

Faeces were collected separately from urine. Usually a single collection was made for the entire experiment.

At the end of the experiment, the animals were anaesthetized with ketamine. Blood was then removed by heart puncture. This was followed by injection of a lethal dose of pentobarbital directly into the heart. Selected tissues were then removed for analysis.

Breath trap solutions of sodium hydroxide were stored at room temperature. Aliquots of plasma, urine and the volatile organic breath traps were assayed immediately. All remaining samples were stored in the dark at -20°C until analysed.

Duplicate aliquots from the volatile organic breath traps, plasma and urine were added to 10 mL of scintillation cocktail. Faeces and large tissues were homogenized in ethanol. After filtration, the residue was washed with water and then oxidized as for tissues. Aliquots of muscle, skin, blood and entire small tissues were oxidised. Some samples of adipose were extracted with cyclohexane, the extract was then analyzed as described for urine. All samples were then analyzed for 14C in a scintillation spectrometer.

Samples were analyzed for parent compound and metabolites using reverse phase HPLC/scintillation spectrometry. Filtered aliquots of urine, volatile organic breath trap solution and plasma were analyzed directly. Tissues were extracted at O°C with cyclohexane, the extracts were filtered through a 0.2µm membrane before injection onto the HPLC. Recoveries of [14C]-cyclohexane from extraction of tissue samples spiked with 1-4 pg/g of [14C]-cyclohexane were as follows liver: 94%; muscle: 93%; skin: 93%.

An alternate method for analysis of cyclohexanone was used for one plasma and one adipose sample in order to confirm the HPLC techniques.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
(91% estimated from total excretion)
Details on distribution in tissues:
Peak at 6 hours after dose reducing to less than 1% after 72 hours. Highest concentrations were in adipose.
Details on excretion:
Exhalation (60-90% of dose) and urine (10-30% of dose). Half-lives (14C) for plasma and tissues, including adipose, were 10-15 hr.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Cyclohexanone & cyclohexanol

Any other information on results incl. tables

While some differences exist between concentrations in plasma and in blood, no trend was observed. The apparent concentration of 14C in plasma varied appreciably from one experiment to the next. Concentrations of cyclohexanone and cyclohexanol were highest at the 2 hr time point. Except for a slight apparent increase in the concentration of cyclohexane during the 6-24 hr interval, concentrations of these substances decreased with increasing time in both sets of experiment. Cyclohexanol was the most abundant metabolite present at 2 and 4 hr.

Seventy-two hours after intravenous administration of [14C]-cyclohexane, the highest concentration of 14C was in adipose and averaged 16 times that in blood. Concentrations of 14C in liver, kidney and skin were also higher than in blood, possibly due to high concentrations of 14C in fat associated with these tissues. Similar results were found in tissues examined 72 hr after oral doses of 200, 1000 and 2000 mg/kg of [14C]-cyclohexane. As doses increased from 200 to 1000 and 2000 mg/kg, the concentrations of 14C in adipose increased relative to concentrations of 14C in blood and other tissues. Adipose-blood ratios for total 14C 72hr after 1000 and 2000 mg/kg oral doses averaged 41 and 47 to 1, respectively. It thus appears that adipose is a "sink" for cyclohexane.

After an oral dose at 200 mg/kg, the highest concentrations of 14C in tissue were in adipose at all time periods studied. Concentrations of 14C were 10-14 times greater in adipose than in blood; 10-17 greater in adipose than in plasma. At 2 hr after dosing, concentrations of 14C in adipose were 103ng-eq/g tissue, approximately 8 times higher than in plasma. The concentrations of cyclohexane in adipose 6, 12, and 24 hr following oral administration of 200 mg/kg were 17,000; 700; and 1400 times that in plasma, respectively.

Concentrations of cyclohexane, cyclohexanone and cyclohexanol were determined in adipose samples taken 72 hr after an intravenous dose of 10 mg/kg of [14C]-cyclohexane, and after an oral dose of 1000 mg/kg of [14C]-cyclohexane and in adipose, liver, muscle and skin samples taken at various times after oral administration of 200 mg/kg of [14C]-cyclohexane. Cyclohexane is the major 14C labelled constituent in adipose, accounting for 79 and 94 percent, respectively, of the 14C in the 72 hr samples taken after 10 mg/kg iv and 1000 mg/kg oral doses and 86 and 94 percent, respectively, of the 14C in the 6 and 24 hr samples taken after a 200 mg/kg oral dose. Low, but measurable, concentrations of compounds eluting from the HPLC with retention times identical to those cyclohexanol and cyclohexanone were also observed.

Much less of the 14C in muscle, liver and skin is attributable to cyclohexane. Cyclohexane accounts for 2% or less of the 14C in muscle, 7% or less of the 14C in liver and 7-18% of the 14C in skin 6 to 24 hr after oral doses of 200 mg/kg . Concentrations of cyclohexanone and cyclohexanol are generally even lower except for muscle, where concentrations of cyclohexane and cyclohexanol are approximately the same. The major metabolites of cyclohexane present in these tissues are not extractable with cyclohexane.

Preliminary experiments showed that less than 0.3% of a 10 mg/kg intravenous dose of [14C]-cyclohexane was excreted in faeces in 48 hr. Cyclohexane itself would not be expected to be excreted in faeces due to its low molecular weight. Therefore, faeces were not routinely analyzed in the remainder of the studies.

The elimination half-lives of total 14C for plasma and tissues, including adipose, were generally about 10-15 hr, with the half life in skin perhaps being slightly longer.

More than 50% of a 10 mg/kg intravenous dose of [14C]-cyclohexane was excreted in breath within 1 hr of dosing; 80% within 1 day of dosing. Small amounts of cyclohexane, 1.3 and 1.4 percent of the dose, were excreted on the second and third day after dosing, respectively. Since animals had to be dosed within the metabolism chamber in order to obtain high recovery of the 14C in the dose, it is likely that there is a large "first pass" volatilization and excretion of cyclohexane via the lungs. No significant amounts of 14CO2 were excreted. Cyclohexane accounted for greater than 98% of the total 14C excreted by breath. Only trace quantities of 14C-labeled material eluted with retention times corresponding to cyclohexanol or cyclohexanone. Approximately 14% of the 10 mg/kg intravenous dose was excreted in urine. More than 80% of this was excreted during the first 24 hr after dosing.

Excretion of orally administered cyclohexane via breath occurred much slower than after intravenous dosing. More than half of each orally administered dose was excreted via breath within 12 hr of dosing. The excessively high recovery of 14C from the 1000 mg/kg dose was likely due to a low estimation of the dose caused by loss of 14C from the dosing solution before it was analyzed. Normalization of the data from this experiment by setting the total recovery to 91% (total recoveries for the other dose levels were 92, 90 and 91% dose) give values for total excretion in breath of 76% of dose and total excretion in urine of 15% of dose. No significant amounts of 14CO2 were excreted following oral administration. More than 90% of the 14C-labeled material excreted in breath following oral doses of [14C]-cyclohexane was parent compound. Small amounts of 14C-labeled material from breath had HPLC retention times identical to those of cyclohexanol and cyclohexanone, but this material accounted for less than 1% of the 14C in breath.

The ratio of the percent dose excreted in breath to that excreted in urine was approximately 2:1 for the 100 and 200 mg/kg oral doses. This ratio increased to 5:1 for the 1000 mg/kg dose and 6.5:1 for the 2000 mg/kg dose. The corresponding ratio for the 10 mg/kg intravenous dose was 6.2:1. Since the overall urinary metabolic profile is the same for all oral dose levels and for the intravenous dose, the differences in the excretion ratio between breath (which is almost all cyclohexane) and urine (which is almost all metabolites) must be related to the capacity for forming the initial cyclohexane metabolite. At the higher dose levels, it appears that the higher blood concentrations of cyclohexane which occur cannot be metabolized rapidly enough to prevent considerable transport of unmetabolized cyclohexane to the lungs.

Only trace quantities of cyclohexane, cyclohexanol or cyclohexanone were excreted in urine. Cyclohexane, if it had been excreted in urine, would most likely have volatilized and have been trapped and included in the 14C-labelled materials excreted in breath. The major urinary metabolites are much more polar than these compounds and elute in three peaks, labelled M-1, M-2 and M-3, the exact nature of these metabolites is not known.

Applicant's summary and conclusion

Executive summary:

After intravenous administration of 10 mg/kg [14C]-cyclohexane to adult male Fischer 344 rats, 54% of the dose was excreted in the breath in the first hr, 80% in 24 hr, and 83% in 72 hr. After oral administration at doses of 2000, 1000, 200 and 100 mg/kg, 78, 76, 62 and 63%, respectively, of the dose was excreted in the breath over 72 hr with the maximum rate of excretion generally occurring from 2-8 hr. In the same experiments

12, 15, 29 and 29%, respectively, of the dose was excreted in urine, compared to 14% excreted in urine following the intravenous dose. No significant excretion of14C in faeces was observed. Cyclohexane accounted for 93-99% of the radiolabel excreted in breath, but less than 0.1% of the radiolabel in the urine. A maximum of 0.04-0.4% of the dose was excreted in breath as cyclohexanone, 0.09-0.6 as cyclohexanol. Less than 0.1% of the dose was excreted in the urine as either of these compounds. Three major urinary metabolites of cyclohexane were observed, all much more polar than cyclohexanone. The nature of these compounds was not ascertained, although they eluted from the high performance liquid chromatographic column in the region typical of conjugates.

 

After an oral dose of 200 mg/kg, concentrations of total14C in plasma were fairly constant over the period from 2-12 hr, but had decreased to about 20% of these values by 24 hr. Cyclohexane was a minor14C-labeled constituent of plasma. Cyclohexanone accounted for about 10% of the total plasma14C 2-4 hr after dosing and from 1-3% of the total plasma14C 6-24 hr after dosing. Concentrations of cyclohexanol in plasma were

generally 2 to 3 times higher than those of cyclohexanone, except that a much larger (8x) difference was observed 6 hr after dosing. At least 5 other metabolites were found in plasma. These metabolites have not been identified.

 

Seventy-two hours after intravenous administration of [14C]-cyclohexane (10 mg/kg), the concentration of 14C in adipose averaged 16 times that in blood. Similar results were found 6, 24 and 72 hr after oral administration of 200 mg/kg of cyclohexane, and values about three times this high were determined after administration of 1000 and 2000 mg/kg oral doses. The half lives of total14C in plasma and tissues, including adipose, were generally about 10-15 hr. 

Cyclohexane accounted for 79-84% of the 14C in adipose, but only 2-18% of the14C in muscle, liver and skin. Small amounts of cyclohexanone and cyclohexanol were present in all of the tissues.