Registration Dossier

Administrative data

Link to relevant study record(s)

Description of key information

In animals, by oral and inhalation routes, cyclohexane is almost completely absorbed and a figure of 100% absorption will be taken into account for any hazard and risk assessment. Dermal absorption of cyclohexane in liquid phase (i.e. by direct contact with liquid) in rats can be estimated as 5%, as concluded in the RAR (2004). Cyclohexane expected to have low potential for bioaccumulation, based on an estimated BCF of 167 calculated using an experimental log Kow of 3.44. 

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
5
Absorption rate - inhalation (%):
100

Additional information

The toxicokinetic profile of cyclohexane including absorption, metabolism, distribution and elimination is considered in this endpoint summary.  As required under Annex VIII Section 8.8.1 this includes assessment of the toxicokinetic behaviour of cyclohexane to the extent that can be derived from the relevant available information.  The primary source is the RAR (2004).  The literature search conducted in support of this application has not resulted in any additional data considered of relevance being sourced.  As detailed in the information gathering requirements (TGD R.3.2), it would serve little to add to the overall assessment by revisiting all of the primary information sources cited in the RAR (2004), and therefore robust and supporting summaries have been prepared for the selected key and supportive studies.  However, other relevant studies have been discussed within the endpoint summary.

Non-human information

The toxicokinetics of cyclohexane have been studied in rat (RTI, 1984), rabbit (Elliott et al., 1959) and mouse (Naruse, 1984).

Male Fisher-344 rats were administered single oral doses by gavage of 100, 200, 1000 or 2000mg [14C]-cyclohexane per kg bodyweight, an additional group of rats received a single intravenous dose of 10 mg [14C]-cyclohexane per kg bodyweight. Exhaled volatiles, urine and faeces were collected at intervals up to 72 hours after dosing. Blood, plasma and selected tissues were retained at termination and analysed for total 14C; metabolite profiles were determined by HPLC.

Female Chinchilla rabbits were administered single oral doses by gavage of 0.3, 100 or 350 - 400mg [14C]-cyclohexane per kg bodyweight in a number of small experiments. Exhaled volatiles, urine, faeces and tissues were retained and analysed for total 14C; metabolite profiles were determined by paper chromatography or derivatisation. In an additional experiment, doses of cyclohexane and putative metabolites were administered individually and the degree of conjugation with glucuronic acid was determined.

Mice were exposed for 1 hour by inhalation to cyclohexane vapour derived from an adhesive; vapour concentrations of 8000, 14000 and 17500ppm were used. Blood concentrations of cyclohexane were determined during and after exposure.

The ability of cyclohexanol to induce the activity of specific male Wistar rat cytochrome (CYP) P450 isozymes was investigated (Espinosa-Aguirre et al., 1996 & 1997). Rats were exposed orally to cyclohexanol (2.5% v/v in drinking water, ad libitum) for 5 days prior to determination of isozyme activity using Western blot analysis.

Absorption

Although absorption was not determined specifically, minimum values can be estimated from the total of the amounts excreted in exhaled volatiles and urine and that retained in tissue. Using this approach, estimates for absorption of cyclohexane after gavage dosing are approximately 91% for rats (100 - 2000mg/kg bodyweight) and 95% for rabbits (0.3 - 400mg/kg bodyweight). A QSAR prediction for dermal absorption of 2.2% was obtained, but was not utilized because observed experimental data was available.

Distribution

Following administration of 200mg [14C]-cyclohexane per kg bodyweight to rats, concentrations of total 14C in whole blood and plasma were similar; there was considerable inter-animal variation in concentrations when the same dose was administered on three separate occasions to different rats. Peak concentrations of total 14C in blood and plasma were attained by 6 - 12 hours after dosing. Concentrations of total 14C in all studied tissues were greatest 6 hours after dosing and were significantly lower by 72 hours after dosing. Tissue residues of total 14C at 72 hours after administration accounted for approximately 0.4% of a dose of 200mg [14C]-cyclohexane per kg bodyweight. Following dosing by either intravenous (10mg/kg) or oral (200, 1000 or 2000mg/kg) routes, the highest concentrations of 14C at 72 hours after dosing were in adipose tissue. The ratios of the concentrations of 14C in adipose to those in blood ranged from approximately 16:1 for the 10mg/kg intravenous and 100mg/kg oral doses to 41:1 and 47:1 for the 1000 and 2000mg/kg oral doses (RTI, 1984).

The tissue distribution of [14C]-cyclohexane was not determined in rabbit tissues; total residues in tissue at termination (3 - 6 days after administration) were approximately 2.5% of the dose (Elliott et al., 1959).

Mice, exposed for 1 hour by inhalation to cyclohexane vapour at concentrations of 8000, 14000 and 17500ppm had peak blood concentrations of cyclohexane of 27, 69 and 122µg/mL respectively; blood concentrations had fallen to 2 - 4% of peak values by 2 hours after the end of exposure (Naruse, 1984).

Metabolism

Following an intravenous dose of 10mg [14C]-cyclohexane per kg bodyweight to rats, 79.5% of the dose was exhaled unchanged during the initial 24 hours after dosing with a further 1.27% and 1.43% exhaled during the 24 - 48 and 48 -72 hour periods respectively. 14C exhaled as either cyclohexanone or cyclohexanol only accounted for a total of 0.22% of the dose over the 0 - 72 hour period after dosing. Following oral administration of 100, 200 or 1000mg/kg, unchanged cyclohexane exhaled during the 72 hour period following dosing accounted for 59.4, 59.8 and 92.1% of the doses respectively. The corresponding values for 14C exhaled as either cyclohexanone or cyclohexanol were 0.09, 0.62 and 0.24% of the dose respectively. The report authors commented that the high value for cyclohexane after a dose of 1000mg/kg may have been due to an error in estimating the dose. No significant amounts of 14CO2 were detected after any of the doses.

Similar urinary metabolite profiles were observed after both intravenous and oral administration and at all oral dose levels. Only trace quantities of cyclohexane, cyclohexanone and cyclohexanol were present, the majority of the 14C was present as four unidentified polar metabolites.

Plasma contained minor amounts of unchanged cyclohexane along with cyclohexanone, cyclohexanol and five unidentified metabolites that accounted for at least half the total 14C present.

Unchanged cyclohexane in adipose accounted for 79 and 94% of the total 14C present 72 hours after the 10mg/kg intravenous and 1000mg/kg oral doses respectively; small amounts of cyclohexanone and cyclohexanol were also detected. The majority of the total 14C present in muscle, liver and skin was not extractable; small amounts of cyclohexane, cyclohexanone and cyclohexanol were detected (RTI, 1984).

In rabbits unchanged [14C]-cyclohexane in exhaled volatiles accounted for 25 - 38% of a dose of 360 - 390mg [14C]-cyclohexane per kg bodyweight; an additional 8 - 10% of the dose was eliminated as 14CO2; after a low dose of 0.3 mg[14C]-cyclohexane per kg bodyweight all exhaled radioactivity (6% of dose) was present as 14CO2. No cyclohexanone or cyclohexanol was detected in exhaled volatiles.

The major urinary metabolites were cyclohexanol accounting for 35 - 50% of the high dose and 60% of the low dose and (±) trans-cyclohexane -1, 2 diol accounting for 3 – 8% of the high dose and 17% of the low dose; both metabolites were present as the glucuronic acid conjugate (Elliott et al., 1959). Oral exposure of rats to cyclohexanol for 5 days resulted in an increase in the activity of CYP2E1 and CYP2B1/B2 but not of CYP1A1/A2 (Espinosa-Aguirre et al., 1996 & 1997).

Excretion

In rats the major route of excretion of 14C was in exhaled volatiles accounting for 83.2, 63.4, 61.8, 96.8* and 78.5% of the dose following 10mg/kg intravenous, 100, 200, 1000 or 2000mg/kg oral doses respectively; the corresponding values for urinary excretion were 13.5, 28.8, 28.6, 19.3* and 12.0% of the dose (* the high values after the 1000mg/kg oral dose may be due to an error in estimating the dose). A preliminary experiment showed that as only minor amounts of 14C ( <0.3% of the dose) were excreted in faeces; samples were not analysed for the remainder of the studies. Elimination half lives for total 14C from plasma and tissues were 10 - 15 hours with a slightly longer value for skin (RTI, 1984).

In rabbits administered 360 - 390mg [14C]-cyclohexane per kg bodyweight, 35 – 47% of the dose was eliminated in exhaled volatiles; at 0.3mg [14C]-cyclohexane per kg bodyweight the corresponding values was 6% of the dose. Urinary excretion of radioactivity at the high and low dose levels was 33 - 56% and 87% of the doses respectively. Faecal excretion of radioactivity (0.1-0.2% of dose) was minimal (Elliott et al., 1959).

Human Information

Human Volunteer Studies

Human volunteer studies involving exposure by inhalation to cyclohexane vapour have been conducted (Mraz et al., 1998; 1999). 

In the initial study, 4 male and 4 female volunteers were exposed by inhalation to cyclohexane vapour (1010mg/m3) for 8 hours. Urine was collected for 72 hours, glucuronide conjugates were hydrolysed and cyclohexanol (CH-ol), cyclohexane-1,2, diol (CH-1,2 diol) and cyclohexane-1, 4, diol (CH-1,4 diol) excretion was determined using gas chromatography. Urinary excretion of CH-ol, CH-1,2 diol and CH-1,4 diol accounted for 0.5, 23.4 and 11.3% of the absorbed dose respectively. Excretion of CH-ol declined rapidly after exposure but elimination of the CH-diols reached maximal values a few hours after exposure and then declined with a half life of approximately 17 hours. No sex difference in metabolite profile or excretion rates was observed. Very low concentrations of cyclohexane and cyclohexanone were detected in urine. Additionally, volunteers were dosed orally with the diols and the urinary excretion monitored. Peak excretion rates occurred within 4 hours; the elimination half lives were15 and 19 hours for the 1,2 and 1,4 diols respectively. The 1,2 diol was excreted predominantly (>95%) as the glucuronide conjugate whereas the 1,4 diol was excreted unconjugated. An in-vitro experiment showed that there is negligible binding of the diols to plasma proteins.

The second study investigated the effects of ethanol ingestion (4 x 14g doses during exposure) on cyclohexane metabolite excretion after inhalation exposure (1000mg/m3) for 8 hours. Urinary excretion of CH-ol, CH-1,2 diol and CH-1,4 diol accounted for 3.1, 15 and 8% of the absorbed dose respectively. The report authors concluded that the diols are less sensitive to ethanol ingestion than CH-ol and should be used for biomonitoring.

Occupational Exposure Monitoring

Several investigators have monitored occupational exposure to cyclohexane. 

Concentrations of cyclohexane in environmental air, alveolar air and blood and urinary excretion of cyclohexanol were measured in shoe factory workers (Perbellini and Brugnone, 1980). Concentrations of cyclohexane in alveolar air (16 - 1929mg/m3) were 78% of those in environmental air (17 - 2484 mg/m3), blood concentrations of cyclohexane (29 - 367µg/L) 4 hours after exposure were 53 - 78% of those in environmental air. Urinary excretion of cyclohexanol accounted for 0.1 - 0.2% of the absorbed dose and was correlated with blood concentrations.

Mutti et al., (1981) investigated lung uptake during exposure (6 hour) and alveolar excretion of cyclohexane during the 6 hours post exposure period in 3 volunteers and 5 workers at a shoe factory. Alveolar retention of cyclohexane was found to be 34% of the inhaled dose corresponding to a lung uptake of 23%. Post exposure alveolar excretion was less than 10% of the total uptake. After high exposures, 40% of the dose was excreted unchanged in exhaled air with an additional 10% present as CO2; after lower exposures, the corresponding values were 10 and 5% respectively. Urinary excretion of cyclohexanol and cyclohexanone accounted for only about 1% of the absorbed dose.

The weighted environmental concentration in the breathing zone over a 4 hour exposure period at the start of work was measured in a group of 43 workers using a personal passive dosimeter (Ghittori et al., 1987). Urine was collected post-exposure and analysed for cyclohexane by gas chromatography. The investigators reported a linear relationship between the weighted environmental concentration and urinary cyclohexane concentrations.

Yasugi et al.(1994) monitored 33 female workers who either applied glue containing cyclohexane or worked in the vicinity of glue application. The geometric mean and highest cyclohexane concentrations measured in environmental air using diffusion samplers were 27 and 274 ppm (93 and 943mg/m3) respectively. There was a significant correlation between atmospheric concentrations of cyclohexane and both concentrations of cyclohexanol in urine and cyclohexane in blood and serum; urinary concentrations of cyclohexanone were less well correlated. Only <1% of the absorbed dose was excreted in urine as cyclohexanol almost exclusively as the glucuronide; the half life was estimated at 5 hours.

Perico et al. (1999) monitored atmospheric concentrations of cyclohexane in shoe and leather factories and the excretion of cyclohexane-1,2 diol and cyclohexane-1,4 diol in the urine of exposed workers. Atmospheric concentrations of cyclohexane were measured throughout the 6 - 7 hour working shift using active personal samplers. Urine samples were collected throughout a working week as follows; Monday pre-shift (n=29), Monday post-shift (n=86), Thursday post-shift (n=70) and Friday pre-shift (n=70); metabolite concentrations were determined by gas chromatography after hydrolysis of conjugates. Urine samples were also obtained from subjects not occupationally exposed to cyclohexane to determine whether background concentrations of the metabolites were present. Individual exposure to cyclohexane ranged from 7 - 617mg/m3 (geometric mean - 60mg/m3). Urinary concentrations of cyclohexane-1,2 diol (geometric means) were 3.1, 7.6, 13.2 and 6.3mg/g creatinine for the Monday pre- and post-shift samples, Thursday post-shift sample and Friday pre-shift samples respectively. The corresponding values for cyclohexane-1,4 diol were 2.8, 5.1, 7.8 and 3.7mg/g creatinine. There was a good correlation between environmental exposure to cyclohexane and Monday post-shift values for the diols but poor correlation with the samples taken on Thursday and Friday. The poor correlation after repeated exposure was attributed to accumulation of the diols during the week (half lives were approximately 18 hours). Workers not occupationally exposed to cyclohexane had urinary concentrations (geometric means) of 0.4 and 1.2mg/g creatinine for cyclohexane-1,2 diol and cyclohexane-1,4 diol respectively.

Additional Data

Cyclohexane was detected but not quantified in 5 samples of human milk collected in different towns and states of the US and analysed for volatile and semi-volatile organic compounds using gas chromatography - mass spectrometry (Pellizzari, 1982).

Additional references cited (not included in IUCLID):

Espinosa-Aguirre, J. J., Rubio, J., Lopez,I., Nosti, R. and Asteinza, J. (1996) Induction of microsomal enzymes in liver of rats treated with cyclohexanol, Mutat. Res., 368, 103 - 107.

Ghittori, S., Imbriani, M., Pezzagno, G. and Capodaglio, E. (1987) The urinary concentration of solvents as a biological indicator of exposure: proposal for the biological equivalent exposure limit for nine solvents,Am.Ind.Hyg. Assoc. J., 48, 786 - 790.

Mraz, J., Galova, E.,Nohova, H., Vitkova, D. and Tichy, M. (1999) Effect of ethanol on the urinary excretion of cyclohexanol and cyclohexanediols, biomarkers of the exposure to cyclohexanone, cyclohexane and cyclohexanol in humans,Scan. J. Work Environ. Health,25, 233 - 237.

Mutti, A., Falzoi, M., Lucertini, S., Cavatorta, A. and Franchini,I.(1981), Absorption and alveolar excretion of cyclohexane in workers in a shoe factory,Journal of Applied Toxicology, 1, 220 - 223.

Naruse, M. (1984) Effects on mice of long-term exposure to organic solvents in adhesives,Nagoya Med. J., 28, 183 - 210.

Pellizzari, E. D., Hartwell, T. D., Harris, B. S. H., Waddell, R. D., Whitaker, D. A. and Erickson, M. D. (1982) Purgeable organic compounds in mother’s milk,Bull. Environ. Contam. Toxicol., 28, 322 - 328.

Perbellini, L. and Brugnone, F. (1980) Lung uptake and metabolism of cyclohexane in shoe factory workers,Int. Arch. Occup. Environ. Health45, 261 - 269.

Perico., A., Cassinelli, C., Brugnone, F., Bavazzano, P., and Perbellini, L. (1999) Biological monitoring of occupational exposure to cyclohexane by urinary 1,2- and 1,4-cyclohexanediol determination,Int. Arch. Occup. Environ. Health, 72, 115 - 120.

Yasugi, T., Kawai, T., Mizunuma, K., Kishi, R., Harabuchi,I., Yuasa, J., Eguchi, T., Sugimoto, R., Seiji, K. and Ikeda, M. (1994) Exposure monitoring and health effect studies of workers occupationally exposed to cyclohexane vapour,Int. Arch. Occup. Environ. Health, 65, 343 - 350.

Discussion on absorption rate:

The toxicokinetics of cyclohexane after dermal exposure have been studied in rat (RTI, 1996; Lyadomi et al., 1998).

In the first study, groups of male and female Fisher-344 rats were exposed dermally to occluded doses of 1 or 100mg/cm2[14C]-cyclohexane for 6 hours; an additional group of rats received a single intravenous dose of 10 mg [14C]-cyclohexane per kg bodyweight. The report authors commented that the low dermal dose was present primarily as vapour while the high dose was present primarily as liquid. Exhaled volatiles, urine and blood samples were collected up to 72 hours after dosing and analysed for total14C. Carcasses were retained at termination and analysed for residues of total14C. Lyadomi et al. (1998) measured blood concentrations of cyclohexane in male WBN/ILA-Ht hairless rats using headspace capillary gas chromatography after dermal exposure for 2 hours to 1mL cyclohexane applied to 3.14cm2skin within an affixed chamber.

Absorption

Absorption of cyclohexane after dermal exposure to 1mg/cm2was 36% (0.06mg/cm2/h) and 60% (0.10mg/cm2/h) in male and female rats respectively and 4% (0.65mg/cm2/h) in both sexes after dermal exposure to 100mg/cm2. The small group sizes and considerable inter-animal variability in absorption prevented an assessment of the relevance of the apparent sex difference at the low dose level.  Lyadomi et al. (1998) did not measure absorption rates but demonstrated that cyclohexane rapidly entered the systemic circulation.

Distribution

Neither study measured tissue distribution of absorbed cyclohexane. In the RTI (1996) study, total 14C residues in carcasses at 72 hours after exposure to [14C]-cyclohexane were <0.4% and <0.1% of doses of 1 and 100mg/cm2 respectively.  Lyadomi et al. (1998) found that blood concentrations of cyclohexane reached a peak of approximately 0.24 µmol/L within 1 hour of the start of exposure and then declined until the end of the 2 hour exposure.

Metabolism

No assessment of the metabolism of cyclohexane was made in either study.

Excretion

Exhaled volatiles accounted for 70% of the radioactivity excreted from the 10mg/kg intravenous dose and 78% and 57% of the radioactivity excreted from the 1 and 100 mg/cm2 dermal doses respectively. Urinary excretion of total 14C represented 29%, 20% and 40% of the excreted radioactivity from the intravenous, low dermal and high dermal doses respectively.

Additional references cited (not included in IUCLID):

Lyadomi, M., Higaki, Y., Ichiba, M., Morimoto, M. and Tomokunii, K. (1998) Evaluation of organic solvent-induced inflammation modulated by neuropeptides in the abdominal skin of hairless rats,Indust. Health, 36, 40 - 51.