Methylcyclohexane

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

Data source

Reference

Reference Type:

publication

Title:

The Metabolism of Methylcyclohexane

Author:

Elliot, T.H. et al.

Year:

1965

Bibliographic source:

Biochem J 95:70-76

Materials and methods

Objective of study:

distribution

excretion

metabolism

Principles of method if other than guideline:

Distribution, metabolism and excretion studies of radiolabelled methylcyclohexane in rabbits following oral administration.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methylcyclohexane

EC Number:

203-624-3

EC Name:

Methylcyclohexane

Cas Number:

108-87-2

Molecular formula:

C7H14

IUPAC Name:

methylcyclohexane

Details on test material:

- Name of test material (as cited in study report): methylcyclohexane
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: ca. 95%
- Impurities (identity and concentrations): ≤ 5% cyclohexane, < 1% toluene
- Specific activity (if radiolabelling): 238 µC/g

Radiolabelling:

yes

Remarks:

14C-methylcyclohexane

Test animals

Species:

rabbit

Strain:

Chinchilla

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Individual metabolism cages: yes

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Methylcyclohexane was administered with water by stomach tube. No further details on dosing solutions/mixtures were reported.

Duration and frequency of treatment / exposure:

Single administration

Doses / concentrations

Remarks:

Doses / Concentrations:
Experiment 1: 2.26 mmol/kg bw (equivalent to ca. 222 mg/kg bw)
Experiment 2: 2.10 mmol/kg bw (equivalent to ca. 206 mg/kg bw)
Experiment 3: 2.41 mmol/kg bw (equivalent to ca. 237 mg/kg bw)

No. of animals per sex per dose / concentration:

1 rabbit per experiment

Control animals:

no

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, expired air and other tissues not further specified
- Time and frequency of sampling: according to the authors, urine was continuously collected for about 58-68 h after dosing until it was found to contain negligible radioactivity. Faeces and expired air were collected in the animal chamber during the same period of time.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: according to the authors, urine was continuously collected for about 58-68 h after dosing until it was found to contain negligible radioactivity.
- From how many animals: 3 animals (samples not pooled)
- Method type(s) for identification: end window counter, paper chromatography and autoradiography


TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): Glucuronides were hydrolysed by treatment with HCl.

Results and discussion

Preliminary studies:

After the administration of methylcyclohexane (432 mg/kg bw) to rabbits, 42% of the dose was excreted as glucuronides and 2% as ethereal sulphate, but there was no increase in mercapturic acid excretion.
Urine after the administration of the hydro carbon had pH 8 and gave an intense naphtharesorcinol reaction. It did not reduce Benedict's solution or give a precipitate with Brady's reagent, and there was no blue colour with 2,6-dichloro-quinonechloroimide in sodium hydrogen carbonate solution or a red colour with ferric chloride, these tests showing the absence of ketones and phenols. From the same urine, methyl (trans-4-methyl-cyclohexyl tri-O-acetyl-β-D-glucosid)uronate was isolated and characterised both by comparison with an authentic specimen and by identification of the hydrolysis products.
Paper chromatography of the fresh urine revealed only one glucuronide spot, whose RF values (0.27 in solvent A and 0.79 in solvent B) were identical with those of trans-4-methylcyclohexyl glucuronide. However, since isomeric 2-, 3- and 4-methyl-cyclohexyl glucuronide have virtually identical RF values, the presence of these other glucuronides could not be excluded. The absence of any significant amount of 1-methylcyclohexyl glucuronide could nevertheless be established (Elliott et al., 1965, Biochem J 95:59 pp.).
The radioautographs revealed the presence of six radioactive spots, only one of which was intense, and this was the only spot (RF 0.57 in solvent I and 0.72 in solvent II) that gave a positive reaction for glucuronides. None of the spots reacted with ninhydrin, indicating that amino acids that might have been formed by transamination were absent. The spray for carboxylic acids gave a positive reaction with two spots, one of which corresponded to the glucuronide metabolite and the other (RF 0.84 in both solvent systems) to hippuric acid. However, the presence of the small amount of free benzoic acid and the possibility of the presence of traces of 1-methylcyclohexanol and cyclohexylmethanol could not be demonstrated conclusively by this method.

Main ADME results

Main ADME results 1

Type:

absorption

Results:

64.7 - 93.2% of dose (mean: 82.4%), calculated from radioactivity distribution in tissues, urine and expired CO2 and methylcyclohexane

Main ADME results 2

Type:

distribution

Results:

tissues (not further specified): 2.8 - 5.9% of dose (mean: 4.2%)

Main ADME results 3

Type:

metabolism

Results:

Major metabolites: glucuronide conjugates of trans-4-methylcyclohexanol (11.6 - 19.4% of dose; mean: 14.7%), cis-3-methylcyclohexanol (8.9 - 15% of dose; mean: 11.5%), and trans-3-methylcyclohexanol (8.5 - 11.9% of dose; mean: 10.5%)

Main ADME results 4

Type:

excretion

Results:

Urine: 54.2 - 77.4% of dose (mean: 65.4%); faeces: 0.4 - 0.7% of dose (mean: 0.53%); expired as CO2: 3.3 - 8.6% of dose (mean: 5.6%); expired as methylcyclohexane: 1.3 - 15.9% of dose (mean: 7.2%)

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Calculated from the reported radioactivity distribution found in tissues, urine and expired CO2 and methylcyclohexane, approx. 64.7, 89.2 and 93.2% methylcyclohexane was absorbed following administration of 222, 206 and 237 mg/kg bw, respectively, most of which was further metabolised within ca. 58-68 h post-dose. The authors noted that the lower overall radioactivity value in the first animal (222 mg/kg bw) was due to an incomplete recovery of radioactivity from the expired air.

Details on distribution in tissues:

About 58-68 h after oral administration of 222, 206 and 237 mg methylcyclohexane/kg bw, 5.9, 3.8 and 2.8% of the radioactivity was respectively recovered in the tissues. No further details on individual organs were reported.

Details on excretion:

About 58-68 h after administration of 222, 206 and 237 mg methylcyclohexane/kg bw, 54.2, 64.5 and 77.4% of the respective dose was excreted in the urine; 4.6, 20.9 and 13.0% was excreted in the expired air (3.3, 5.0 and 8.6% as CO2 and 1.3, 15.9 and 4.4% as parent compound, respectively), and 0.5, 0.4 and 0.7% in the faeces, respectively. The authors pointed out that the recovery of radioactivity from the expired air was incomplete in the first experiment (animal given 222 mg/kg bw).

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The major metabolites found in the urine were the glucuronide conjugates of trans-4-methylcyclohexanol (11.6-19.4% of the dose), cis-3-methylcyclohexanol (8.9-15%), and trans-3-methyl-cyclohexanol (8.5-11.9%). Minor metabolites included glucuronides of cis-4-methylcyclohexanol (2.0-2.8%) and of cis- and trans-2-methyl-cyclohexanol (0.3-0.7% and 1.1-1.2%, respectively). No 1-methylcyclohexanol was found. Small amounts of cyclohexylmethanol (≤ 0.3%) and free and conjugated benzoic acid (1.6-2.2%; about 0.5 and 1.5% being free benzoic and hippuric acid, respectively) suggested some minor aromatisation of the cyclohexane ring via hydroxylation and carboxylation of the methyl group.

Any other information on results incl. tables

Table 1. Distribution of ¹⁴C after a single dose of ¹⁴C-methylcyclohexane in rabbits.

 

	Experiment 1	Experiment 2	Experiment 3	Mean values
Dose (mg/kg bw)	222	206	237	222
Radioactivity found (% of dose) in:
Expired air as CO2	3.3	5.0	8.6	5.6
Expired air as methylcyclohexane	1.3	15.9	4.4	7.2
Tissues	5.9	3.8	2.8	4.2
Faeces	0.5	0.4	0.7	0.5
Urine	54.2	64.5	77.4	65.4
Total radioactivity accounted for:	65.2*	89.6	93.9	82.9

 

* The low value was due to incomplete recovery of radioactivity from the expired air.

 

Table 2. Metabolites of ¹⁴C-methylcyclohexane in the urine of rabbits after hydrolysis.

 

	Experiment 1	Experiment 2	Experiment 3	Mean values
Dose (mg/kg bw)	222	206	237	222
Duration of experiment (h)	60	58	68	62
Radioactivity found (% of dose) in metabolite sought:
1-Methylcyclohexanol	-	-	-	-
(±)-cis-2-Methylcyclohexanol	0.3	0.5	0.7	0.5
(±)-trans-2-Methylcyclohexanol	1.1	1.2	1.2	1.2
(±)-cis-3-Methylcyclohexanol	8.9	10.5	15.0	11.5
(±)-trans-3-Methylcyclohexanol	8.5	11.1	11.9	10.5
cis-4-Methylcyclohexanol	2.8	2.4	2.0	2.4
trans-4-Methylcyclohexanol	13.1	11.6	19.4	14.7
Cyclohexylmethanol	< 0.2	0.3	< 0.3	≤ 0.3
(±)-2-Methylcyclohexanone*	0	0	0	0
(±)-3-Methylcyclohexanone*	0	0	0	0
4-Methylcyclohexanone*	0	0	0	0
Cyclohexanecarboxylic acid	0	0	0	0
Total benzoic acid	1.6	1.8	2.2	1.9
Cyclohexanol**	2.4	2.6	-	2.5
Sum of radioactivities of metabolites sought in the urine	39.1	42.0	52.7	44.6
Total radioactivity in urine	54.2	64.5	77.4	65.4

 

* The hydrolysis procedure was omitted.

** Cyclohexanol is derived from cyclohexane contained in the ¹⁴C-methylcyclohexane sample.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
In this study, the kinetics of 14C-labelled methylcyclohexane was investigated in rabbits given single oral doses of approx. 206-237 mg/kg bw. About 58-68 h after administration, 65.4% of the dose was excreted in the urine, 12.8% in expired air (5.6% as CO2, 7.2% as methylcyclohexane), 0.5% in the faeces; 4.2% remained in the carcass. In a separate experiment, 42 and 2% of the dose were found to be excreted as glucuronide and sulphate conjugates, respectively, in rabbits given ca. 432 mg/kg bw.
The major metabolites found in the urine were the glucuronide conjugates of trans-4-methylcyclohexanol (14.7% of the dose), cis-3-methylcyclohexanol (11.5%), and trans-3-methyl-cyclohexanol (10.5%). Minor metabolites included glucuronides of cis-4-methylcyclohexanol (2.4%) and of cis- and trans-2-methyl-cyclohexanol (0.5% and 1.2%, respectively). No 1-methylcyclohexanol was found. Small amounts of cyclohexylmethanol (≤ 0.3%) and free and conjugated benzoic acid (1.9%; about 0.5 and 1.5% being free benzoic and hippuric acid, respectively) suggested some minor aromatisation of the cyclohexane ring via hydroxylation and carboxylation of the methyl group.