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Description of key information

The available experimental data on the toxicokinetic behaviour of methylcyclohexane demonstrate that the substance is readily and almost completely absorbed by the oral and inhalation routes. For the dermal route, a low to moderate absorption is anticipated from its physico-chemical properties (molecular weight, water solubility, octanol/water partition coefficient and vapour pressure). 
Following absorption, methylcyclohexane is readily distributed to all tissues showing a preference to adipose tissues, but without evidence of bioaccumulation.
The metabolism of methylcyclohexane consists of hydroxylation of the alicyclic ring resulting in the formation of isomers of methylcyclohexanol and methylcyclohexan-diol. Hydroxylation is followed by conjugation of the alcohols to glucuronides (and probably also sulphates) for excretion via the urine.
The main routes of excretion for methylcyclohexane are via the lung in the expired air (either unchanged or as CO2) and in urine as conjugated metabolites.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
50
Absorption rate - inhalation (%):
100

Additional information

Justification for read-across approach

In order to fulfil the toxicological information requirements established in Annexes VII-X, Section 8, of Regulation (EC) No 1907/2006, data from the structurally similar substance cyclohexane (CAS No. 110-82-7) was used for assessment of the endpoints acute toxicity by dermal route, skin sensitisation, in vitro gene mutation in mammalian cells, two-generation reproductive toxicity and pre-natal developmental toxicity in an analogue approach. Following the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, common functional groups and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals, are two criteria for justifying substance similarity and ultimately the read-across approach itself.

Methylcyclohexane and cyclohexane share the 6-membered saturated alicyclic ring of cyclohexane as common molecular structure. It is thus likely that both substances will undergo similar chemical and biological reactions related to the cycloalkane structure. For the purpose of justification of the read-across approach, available information on the toxicokinetic behaviour of cyclohexane is discussed in parallel to that of methylcyclohexane in order to assess similarities, in particular regarding metabolism as a biological process resulting in structurally similar chemicals.

A detailed justification for the read-across approach is provided in the technical dossier (see IUCLID Section 13).

Toxicokinetics

Four studies are available in which one or more toxicokinetic parameters of methylcyclohexane have been investigated, allowing the assessment of the toxicokinetic behaviour of the substance in conjunction with the relevant available information on physico-chemical and toxicological properties according to “Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance” (ECHA, 2008).

Absorption

Methylcyclohexane is a volatile liquid with vapour pressure of 6.18 kPa (at 25 °C), molecular weight of 98.2, water solubility of 14 mg/L (at 25 °C) and Log Pow of 3.88. These values are within ranges suggesting that methylcyclohexane is likely to be readily absorbed via the oral and inhalation route, while dermal absorption is likely to be low to moderate, as discussed below. The assumption of oral and inhalative absorption is confirmed by the available short- and long-term toxicity studies by these routes, as well as by the available studies on toxicokinetic parameters allowing a (semi-)quantitative assessment of absorption.

Similar conclusions can be drawn for cyclohexane based on vapour pressure (10.3 kPa at 20 °C), molecular weight (84.16), water solubility (58 mg/L at 25 °C), Log Pow (3.44) and experimental animal and human data (EC-ECB, 2004).

- Oral

In a study by Elliot et al. (1965) in rabbits, approx. 89.2, 64.7, and 93.2% methylcyclohexane (mean: 82.4%) was absorbed following administration of 206, 222, and 237 mg/kg bw, respectively, as calculated from the radioactivity distribution in tissues, urine and expired unchanged methylcyclohexane and CO2. Most of the applied dose was further metabolised within ca. 58-68 h. The authors noted that the lower overall radioactivity value in the animal given 222 mg/kg bw was due to an incomplete recovery of radioactivity from the expired air. Further studies on urine metabolites in rats and rabbits (Parnell et al., 1988; Treon et al., 1943a) as well as the available repeated dose oral toxicity studies provide evidence of systemic absorption via the oral route.

Based on the study by Elliot et al. (1965) the oral absorption rate of methylcyclohexane is ≥ 80%. For the purpose of chemical safety assessment, a value of 100% is assumed.

The toxicokinetic behaviour of cyclohexane following oral administration has been investigated in rats and rabbits.

Seventy-two hours after single oral administration of radiolabelled cyclohexane (100-2000 mg/kg bw) to rats, most of the radioactivity (63-78%) was recovered in the expired air as unchanged cyclohexane and in the urine (29-12%) mainly as conjugated and free metabolites (RTI, 1984, as cited in EC-ECB, 2004). A dose-dependent increase in the amount of expired cyclohexane and simultaneous decrease in urinary excretion was apparent. No significant faecal excretion was observed.

In rabbits given a single dose of radiolabelled cyclohexane at 100-390 mg/kg bw and monitored for 2-6 days, the main routes of excretion were expired air and urine at similar proportions (35-47% and 46-55%, respectively), without significant faecal excretion (Elliot et al., 1959, as cited in EC-ECB, 2004). Unchanged cyclohexane accounted for most of the radioactivity recovered in the expired air (25-38% of dose), the remaining being 14CO2. In the same study, the authors reported that, when cyclohexane was administered at a lower dose (0.3 mg/kg bw), the main route of excretion was in urine (87% in 4 days), while much less radioactivity was recovered as expired 14CO2 (5.5%), unchanged cyclohexane not being detected.

Both studies indicate that cyclohexane is rapidly and almost completely absorbed (≥ 90%) after oral administration. Furthermore, at higher dose levels a significant proportion of cyclohexane is expired unchanged, which could be explained by a preferential partitioning to alveoli rather than to blood, where it has low solubility (US EPA, 2003).

- Inhalation

Methylcyclohexane was determined in selected tissues of rats after 1, 2 and 3 days of exposure to 400 mg/m³ for 12 h/day (Zahlsen et al., 1992). Methylcyclohexane could be found in all examined tissues (see Distribution), thus providing evidence of systemic absorption. In rabbits exposed to methylcyclohexane by inhalation, absorption was indicated by metabolite excretion in the urine as assessed by a decrease in inorganic sulphates and an increase in glucuronic acid excretion (Treon et al., 1943 b). The reported data, however, do not allow quantitative assessment of absorption.

The toxicity of methylcyclohexane following inhalation exposure has been extensively investigated (see Sections Acute toxicity and Repeated dose toxicity). Based on the reported systemic effects (narcosis) mainly observed at high concentrations, there is sufficient evidence for rapid absorption by inhalation.

Together with the available information on oral absorption, the rate of absorption of methylcyclohexane by inhalation is expected to be high and a value of 100% is assumed for the purpose of chemical safety assessment.

Absorption of cyclohexane via the lungs has also been described in rats and rabbits.

Cyclohexane was likewise investigated in the studies by Zahlsen et al. (1992) and Treon et al. (1943b) yielding results similar to those of methylcyclohexane.

A number of studies have been published on the absorption, metabolism and excretion of cyclohexane in humans; most of them are occupational monitoring studies focusing on inhalation exposure to cyclohexane used as a solvent in the Italian shoe industry during the late 1970's (reviewed in US EPA, 2003 and EC-ECB, 2004). In all cases, the results of the studies demonstrated that cyclohexane is rapidly absorbed via the lung by humans. Two of the studies provided quantitative data on absorption, albeit yielding different results. In one study with 8 subjects, 23% of the inhaled cyclohexane was absorbed by the lungs (Mutti et al., 1981), while the other study involving 22 subjects showed absorption rates between 40 and 60% (Perbellini and Brugnone, 1980).

Both animal and human data thus indicate rapid absorption of cyclohexane by inhalation. Based on human data, the absorption rate of cyclohexane via inhalation is expected to be moderate to high.

- Dermal

There are no studies available in which toxicokinetic parameters of methylcyclohexane have been investigated after dermal exposure. Therefore, the assessment of dermal absorption is conducted on a qualitative basis taking into account the available information on the physico-chemical and toxicological characteristics of the substance.

The molecular weight (MW = 98.2) and octanol/water partition coefficient (Log Pow = 3.88) of methylcyclohexane favour dermal absorption. Moreover, methylcyclohexane, being a solvent, has skin defatting properties and is classified as a skin irritant Category 2 according to Annex VI of Regulation (EC) No. 1272/2008. However, water solubility (14 mg/L) and vapour pressure (6.18 kPa at 25 °C) are within ranges anticipating a low to moderate rate of dermal absorption. The dermal permeability coefficient (Kp) of methylcyclohexane can be calculated from Log Pow and MW applying the following equation described in US EPA (2004):

log(Kp) = -2.80 + 0.66 log Pow – 0.0056 MW

The Kp of methylcyclohexane is thus 0.167 cm/h. Considering its water solubility (0.014mg/cm³), the dermal flux of methylcyclohexane is estimated to be ca. 0.002 mg/cm²/h.

Taking all this information into account, the dermal absorption potential of methylcyclohexane is expected to be low to moderate.

This notion is supported by information available on the dermal absorption of the structurally similar substance cyclohexane.

The physico-chemical properties of cyclohexane also lead to the assumption of a low to moderate absorption through the skin, since they fall within the same ranges as those of methylcyclohexane. Furthermore, cyclohexane has also skin defatting properties and is likewise classified as a skin irritant Category 2 according to Annex VI of Regulation (EC) No. 1272/2008.

In rats exposed to cyclohexane for 6 h at 1 mg/cm² (vapour) and 100 mg/cm² (liquid), dermal absorption rates of 0.06-0.1 mg/cm² h and 0.65 mg/cm² h, respectively, have been reported (RTI, 1996, as cited in EC-ECB, 2004). These values correspond to 40-60% and 4% of the total applied doses, respectively. Thus, the dermal absorption of cyclohexane in the vapour phase appears to be 10 times higher of that in the liquid.

In summary, based on physico-chemical properties as well as supporting information from the structurally similar substance cyclohexane, the rate of dermal absorption of methylcyclohexane is low to moderate. For the purpose of chemical safety assessment, 50% absorption through the skin is considered in a worst case approach, taking into account that, in general, dermal absorption will not be higher than oral or inhalation absorption (ECHA, 2010).

Distribution

Zahlsen et al. (1992) investigated the inhalation kinetics of C6 to C10 aliphatic, aromatic and naphthenic hydrocarbons in rats after a 3-day inhalation exposure. Cyclohexane and methylcyclohexane served as the model substances for C6 and C7 naphthenic hydrocarbons, respectively. Groups of animals were exposed to 100 ppm (ca. 343.5 and 400 mg/m³, for cyclohexane and methylcyclohexane, respectively), 12 h/day, for 1, 2 and 3 days. Test substance concentrations were determined in blood, brain, liver, kidney, and perirenal fat immediately after each exposure as well as 12 hours following the final exposure.

Methylcyclohexane concentrations in blood and organ samples showed no significant pattern of increase or decrease during the 3 -day exposure period, suggesting the achievement of a steady state level. During the 3-day exposure period, the highest methylcyclohexane concentrations were found in the perirenal fat tissue (356-550 µmol/kg), followed by the kidney (94.7-127.7 µmol/kg), brain (44.4-47.2 µmol/kg), liver (30.1-32.7 µmol/kg), and blood (5.8-6.4 µmol/kg). After the 12-hour recovery period following the last exposure, concentrations decreased to 0.1 µmol/kg in blood (-98.4%), 0.5 µmol/kg in brain (-98.9%), 0.5 µmol/kg in liver (-98.4%), 2.9 µmol/kg in kidney (-97.4%) and 231 µmol/kg in perirenal fat (-49.3%).

The results of cyclohexane determination in tissues during and after the exposure period were very similar to those of methylcyclohexane. Thus, the highest cyclohexane concentrations were also found in the perirenal fat tissue (417-482 µmol/kg), followed by the kidney (86.5-100.1 µmol/kg), brain (31.7-34.7 µmol/kg), liver (22.3-26.4 µmol/kg), and blood (4.0-4.4 µmol/kg). After the recovery period, concentrations were decreased by 97.6% in blood, 94% in brain, 97.9% in liver, 98.6% in kidney and 63% in perirenal fat.

In another study, about 58-68 h after a single oral administration of 206, 222 and 237 mg 14C-methylcyclohexane/kg bw, 3.8, 5.9 and 2.8% (mean 4.2%) of the radioactivity was respectively recovered in the tissues (Elliot, 1965). No further details on individual organs were reported.

Taken together, the available data indicate that both methylcyclohexane and cyclohexane are readily distributed to all tissues and, as expected for a non-polar organic compound, both substances partition preferentially to lipid-rich tissues (US EPA, 2003; EC-ECB, 2004).

Accumulation

In rabbits given single oral doses of 206-237 mg/kg bw, 59.3-91.1% (mean 78.7%) of the dose was excreted in the urine, in expired air and in the faeces about 58-68 h after administration (Elliot et al., 1965; see also Excretion). The low value of 59.3% was due to incomplete recovery of radioactivity from the expired air. Thus, the mean percentage of the dose excreted 60 h post-dose is likely to be higher (around 90%).

In the study by Zahlsen et al. (1992), cyclohexane and methylcyclohexane concentrations measured in blood, brain, liver, kidney and perirenal fat of rats appeared to have reached a steady state level already within 12 h of exposure to 343.5 and 400 mg/m³, respectively, since no significant pattern of increase or decrease was observed during the 3-day exposure period. Cyclohexane and methylcyclohexane concentrations were reduced by ≥ 94 and 97%, respectively, in tissues of rats held for a 12-hour recovery period following the last exposure. An exception was the methylcyclohexane and cyclohexane concentration in perirenal fat, which was reduced by 49.3 and 63%, respectively.

Based on the results of these studies, both methylcyclohexane and cyclohexane are rapidly eliminated from the body after oral and inhalation exposure. Due to their lipophilic properties, both substances show a tendency to partition to fatty tissue and elimination is slower. Nevertheless, the available data indicate a very short overall biological half-life, and therefore no bioaccumulation potential of methylcyclohexane and cyclohexane is anticipated.

Metabolism

About 60 h after a single oral administration of 206-237 mg methylcyclohexane/kg bw, the major metabolites found in the urine of rabbits were the glucuronide conjugates of trans-4-methylcyclohexanol (11.6-19.4% of the dose; mean 14.7%), cis-3 -methylcyclohexanol (8.9-15%; mean 11.5%), and trans-3-methyl-cyclohexanol (8.5-11.9%; mean 10.5%) (Elliot et al., 1965). 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.

Parnell et al. (1988) investigated the metabolites from the urine of male Fischer 344 rats collected during 48 h following a single oral administration of methylcyclohexane at 800 mg/kg bw. Urinary metabolites identified after glucuronidase/sulphatase hydrolysis included cyclohexylmethanol, trans-3-methylcyclohexanol, trans-4-methylcyclohexanol, cis-2-hydroxy-cis-4-methylcyclohexanol, cis-2-hydroxy-trans-4-methylcyclohexanol, and trans-2-hydroxy-cis-4-methylcyclohexanol in relative abundances of 10.1:2.0:1.0:2.1:15.7:23.4 as determined by gas-liquid partition chromatography. No cyclohexanecarboxylic acid was found. The results of the study suggest that metabolism of the ring structure (dihydroxylation) is strongly favoured in comparison with metabolism of the methyl group.

The hydroxylation pattern of methylcyclohexane was investigated in an in vitro system (Frommer et al., 1970). In the presence of NADPH, oxygen and liver microsome of phenobarbital-treated (80 mg/kg bw/day, for 3 days) rats, mice, rabbits or guinea pigs, methylcyclohexane was hydroxylated to all its isomeric alcohols: 1-methylcyclohexanol, cis-trans-2-methylcyclohexanol, cis-trans-3-methylcyclohexanol, cis-trans-4-methylcyclohexanol and hydroxymethylcyclohexane. The hydroxylation pattern was not significantly affected in rats pre-treated with phenobarbital when compared with untreated controls. In all cases, incubations with microsomes of different species resulted in the formation of cis-trans-3-methylcyclohexanol as the major hydroxylation product (43.4-58.2% of the isomeric alcohols determined in the 4 animal species).

For cyclohexane, animal studies indicate that cyclohexanol is its main metabolite, and lesser amounts of cyclohexanone and 1,2-cyclohexanediol have also been identified.

In a study by Elliott et al. (1959, as cited in US EPA, 2003), the quantitative metabolism of single gavage doses of cyclohexane (0.3 to 390 mg/kg) administered rabbits was investigated. The metabolites detected in urine were mainly cyclohexanol and cyclohexanone as well as small amounts of trans-1,2-cyclohexane-diol. All metabolites were excreted as glucuronides.

In another study, cyclohexanol and cyclohexanone were identified as cyclohexane metabolites in the urine of orally exposed rats (RTI, 1984 as cited in US EPA, 2003 and EC-ECB, 2004).

In humans, cyclohexanol (and its glucuronide) has also been identified as a cyclohexane metabolite. However, it appears that the main metabolic pathway primarily results in the formation of 1,2- and 1,4-cyclohexane-diols, which are then excreted unchanged (1,4-cyclohexane-diol) and in glucuronide form (1,2-cyclohexane-diol) (EC-ECB, 2004). Thus, it seems that the major difference between animals and humans is the formation of cyclohexanol in the first and cyclohexane-diol in the latter. In both animals and humans, oxidation to cyclohexanone is very limited.

In summary, hydroxylation of the ring structure represents the main and common step in both methylcyclohexane and cyclohexane Phase I metabolism, resulting in the formation of methylcyclohexanol (mainly at position 3 and 4) and cyclohexanol, respectively. Further hydroxylation results in the formation of methylcyclohexane-diols (mainly at positions 2 and 4) and 1,2-/1,4-cyclohexane-diols. Differences in metabolism comprise limited hydroxylation at the methyl rest of methylcyclohexane and oxidation of cyclohexanol to cyclohexanone. While there are indications that cyclohexane-diols are the main metabolites in humans, no corresponding data are available for methylcyclohexane.

Phase II metabolism of both methylcyclohexane and cyclohexane primarily consists in conjugation of their hydroxylated metabolites to glucuronides; however, sulphate conjugation may occur at high dose levels.

Excretion

Elliot et al. (1965) determined that about 58-68 h after administration of 222, 206 and 237 mg methylcyclohexane/kg bw, 54.2, 64.5 and 77.4% (mean 65.4%) of the respective dose was excreted in the urine; 4.6, 20.9 and 13.0% (mean 12.8%) was excreted in the expired air (3.3, 5.0 and 8.6% (mean 5.6%) as CO2 and 1.3, 15.9 and 4.4% (mean 7.2%) as parent compound, respectively), and 0.5, 0.4 and 0.7% (mean 0.5%) 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).

Following oral and inhalation exposure, cyclohexane is mainly excreted via the lung (unchanged and as CO2) and in urine as conjugated metabolites; minimal amounts are excreted in the faeces (US EPA, 2003; EC-ECB, 2004). For the oral route, it appears that the proportion of unchanged cyclohexane in the expired air increases with dose, with a concomitant decrease in urinary excretion (RTI, 1984, as cited in EC-ECB, 2004). Accordingly, low oral dose levels of cyclohexane are largely metabolised and excreted via the urine (Elliot, 1959, as cited in EC-ECB, 2004). There is evidence in humans that cyclohexane can be excreted via the milk (RTI, 1980, as cited I EC-ECB, 2004).

Taken together, methylcyclohexane and cyclohexane are eliminated as unchanged compounds or CO2 via the lungs and their metabolites are mainly conjugated to glucuronides and excreted in the urine. Faecal excretion is not significant. The proportion of excretion in the expired air and via the urine is likely to be dose-dependent.

Conclusions for read-across approach

The available experimental data on the toxicokinetic behaviour of methylcyclohexane and cyclohexane demonstrate that both substances are readily and largely absorbed by the oral and inhalation routes. For the dermal route, a low to moderate absorption is anticipated from the (similar) physico-chemical properties of both substances. This notion has been experimentally confirmed for cyclohexane.

Following absorption, both substances are readily distributed to all tissues showing a preference to adipose tissues, but without evidence of bioaccumulation.

Methylcyclohexane and cyclohexane share common Phase I and II metabolic pathways. These consist of hydroxylation of the alicyclic ring resulting in the formation of isomers of methylcyclohexanol/methylcyclohexan-diol and cyclohexanol/cyclohexan-diol, respectively. Hydroxylation is followed by conjugation of the alcohols to glucuronides (and probably also sulphates) for excretion via the urine.

The main routes of excretion for both methylcyclohexane and cyclohexane are via the lung in the expired air (either unchanged or as CO2) and in urine as conjugated metabolites.

The metabolites of methylcyclohexane and cyclohexane are not common breakdown products in terms of degradation. However, hydroxylation and subsequent glucuronidation of both substances result in structurally similar chemicals (cycloalcohols and their glucuronides). Therefore, the common biological process of metabolism demonstrates similarity between methylcyclohexane and cyclohexane and justifies the read-across approach for assessment of toxicological properties.

Reference not in IUCLID

ECHA (2008). Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance. Source: European Chemicals Agency, http://echa.europa.eu/

ECHA (2010). Guidance on information requirements and chemical safety assessment Chapter R.8: Characterisation of dose [concentration]-response for human health. Source: European Chemicals Agency, http://echa.europa.eu/

Elliot TH, Parke DV, WilliamsRT (1959). Studies in detoxication. 79. The metabolismof cyclo[14C]hexane and its derivatives. Biochem.J. 72, 193-200, (cited inthe 1991 HSE ToxicityReview (25) on Cyclohexane).

EUROPEAN COMMISSION - European Chemicals Bureau (EC-ECB) (2004). European Union Risk Assessment Report: Cyclohexane; CAS No: 110-82-7; EINECS No: 203-806-2. European Chemical Bureau - Institute for Health and Consumer Protection.

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

Perbellini L and Brugnone F (1980). Lung uptake and metabolism of cyclohexane in shoe factory workers. Int. Arch. Occup. Environ. Health 45, 261-269.

Research Triangle Institute (1980). Acquisition and Chemical Analysis of Mother's Milk for Selected Toxic Substances. Prepared by RTI for Environmental Protection Agency (Washington DC), December 1980.

Research Triangle Institute (1984). Adsorption, Distribution, Metabolism and Excretion of Cyclohexane. Project Report No. 5 submitted to the National Institute of Environmental Health Sciences. Contract No NO1-ES-1-5007 EPA-OTS Doc 40-8423127. NTIS/OTS0527475.

RTI (1996). Dermal Absorption of [14C] Cyclohexane in Fisher 344 Rats. Comparison of the Disposition of Dermally and Intravenously Administered [14C]Cyclohexane.

US Environmental Protection Agency (EPA) (2003). Toxicological review of cyclohexane (CAS No. 110-82-7) : in support of summary information on the Integrated Risk Information System (IRIS). Washington, DC. EPA 635/R-03/008.

US EPA (2004).  Risk Assessment Guidance for Superfund (RAGS), Volume I: Human Health Evaluation Manual (Part E, Supplemental Guidance for Dermal Risk Assessment) Interim. http://www.epa.gov/oswer/riskassessment/ragse/index.htm