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Administrative data

Key value for chemical safety assessment

Additional information

Genetic toxicity in vitro

- Gene mutation in bacteria

A bacterial gene mutation assay was conducted with methylcyclohexane following a protocol compliant with OECD guideline 471 and under GLP conditions (NITE, 2001). The preincubation procedure was performed with Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 as well as with Escherichia coli WP2 uvrA in the absence and in the presence of a metabolic activator (phenobarbital- and 5,6-benzoflavone-induced rat liver S9 mix). In two preliminary dose-finding studies, cytotoxic effects were observed at concentrations as low as 2.29 µg/plate (TA 100 and TA 1535) without metabolic activation and 20.6 µg/plate (TA 100, TA 1535 and TA 1537) with metabolic activation. Therefore, the main study was conducted with concentrations ranging from 0.0977 to 25 µg/plate in the absence of S9 mix, and from 0.781 to 200 µg/plate in the presence of S9 mix. Six concentrations were selected according to the strain specific cytotoxicity. Methylcyclohexane inhibited cell growth in all strains at the respective highest doses (3.13-25 µg/plate without S9 mix; 25-200 µg/plate with S9 mix). No increase in revertant colonies was observed in any strain either in the presence or absence of metabolic activation. The positive and negative (vehicle: acetone) controls included in the experiments showed the expected results.

Under the conditions of this study, methylcyclohexane did not induce gene mutation in bacteria.

- Chromosome aberrations

Methylcyclohexane was evaluated in an in vitro mammalian chromosome aberration test conducted in accordance with GLP and OECD guideline 473 (NITE, 2001). Chinese hamster lung (CHL/IU) cells were treated with the test substance at 245, 490 and 980 µg/L with and without metabolic activation (S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver). In the first experiment, cells were incubated with the test substance for 6 h (short-term treatment) in the presence and absence of metabolic activation, and harvested after 18 h incubation in cell culture medium (24 h after the onset of the treatment). In the second experiment (continuous treatment), cells were incubated with the test substance for 24 and 48 h without metabolic activation.

No clear cytotoxic effect was observed as assessed both in preliminary growth inhibition tests (short-term and continuous treatment) as well as in the main experiments.

No statistically significant increase in chromosomal aberration was observed at any dose. The incidence of aberrant cells in the cultures treated with the test substance was similar to that of the negative controls in the two experiments (short-term and continuous treatment). For both tests, the incidence of aberrant cells in the negative and positive controls was within the range of historical laboratory data.

Under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test performed in Chinese hamster lung (CHL/IU) cells.

- Gene mutation in mammalian cells

There are no studies available in which methylcyclohexane has been tested for the induction of gene mutations in mammalian cells. In order to fulfil the standard information requirements set out in Annex VII, 8.4, read-across from the reference substance cyclohexane (CAS No. 110-82-7) is conducted in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Cyclohexane was tested for its ability to induce forward mutations in a Mouse Lymphoma Assay conducted following a protocol equivalent to OECD guideline 476 (Pence, 1982d). Mouse lymphoma L5178Y cells were incubated with the test material at 8, 12, 17, 24, 34, 50, 70 and 100 µg/mL for 4 h in the presence and absence of a metabolic activation system (S9 mix). The maximum concentration of 100 µg/mL was selected based on a preliminary toxicity test (65% growth inhibition in the presence of metabolic activation). Untreated, solvent (DMSO) and positive controls (ethylmethanesulphonate, -S9 mix; 3-methylcolanthrene, +S9 mix) were included in the test.

No cytotoxicity and no increase in mutant frequency was observed at any concentration without metabolic activation. In the presence of S9 mix, cytotoxic effects were observed at the highest concentration and a 2- to 2.5-fold increase in mutant frequency was noted at 8, 24 and 70 µg/mL. However, the increases in forward mutations were not dose-related and therefore the criteria for a positive result were not met. Therefore, the test was considered negative in the limits of the range tested.

In another mouse lymphoma assay, cyclohexane was tested negative for the induction of forward mutations at concentrations as high as 7800 µg/mL with and without metabolic activation (EC-ECB, 2004).

In summary, the available in vitro studies do not indicate that methylcyclohexane and the structurally similar reference substance cyclohexane have genotoxic properties.

 

Justification for read-across approach

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests", which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, 1.5, of Regulation (EC) No 1907/2006, whereby physicochemical, toxicological and ecotoxicological properties may be predicted from data for reference substance(s) by interpolation to other substances on the basis of structural similarity, cyclohexane (CAS No. 110-82-7) is selected as reference substance for assessment of ecotoxicological and toxicological endpoints, for which no methylcyclohexane data are available.

Similarity is based on:

(1) common functional group: Methylcyclohexane and cyclohexane share a 6-membered saturated alicyclic ring as a common molecular structure and are therefore allocated to the group of monocyclic hydrocarbons (cycloalkanes);

(2) common precursors and likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals: Methylcyclohexane and cyclohexane do not share a common precursor; however, both substances are produced by hydrogenation of the corresponding aromatic compounds toluene (see Section Manufacture and Use) and benzene (EC-ECB, 2004), respectively, which are in turn structurally similar substances. In general, methylcyclohexane and cyclohexane are likely to undergo the same chemical reactions characteristic of cycloalkanes, e.g. combustion to CO2 and H2O and halogenation (Breitmeier and Jung, 2005). The assessment of toxicokinetic behaviour indicates that both substances share a common main metabolic pathway, namely mono- and dihydroxylation of the alicyclic ring resulting in the respective isomers of methylcyclohexanol/-diol and cyclohexanol/-diol, followed by conjugation to the corresponding glucuronides (see Toxicokinetics); and

(3) constant pattern in the changing of the potency of the properties between substances: For methylcyclohexane and cyclohexane, the constant pattern is characterized by similarities in the potency of properties.

- Physicochemical properties:

Both substances show overall similar physico-chemical properties, being liquids, moderately volatile, lipophilic and slightly soluble in water. (see Section 4, Physical and chemical properties and EC-ECB, 2004).

- Environmental fate and ecotoxicological profile:

Methylcyclohexane and cyclohexane show similar properties in environmental fate and ecotoxicological profile: both substances are volatile, not bioaccumulative and show corresponding adsorption values (see section 5, Environmental fate and pathways and EC-ECB, 2004). Furthermore the available experimental data demonstrate that both substances exhibit a similar ecotoxicity profile. Both are acute very toxic to aquatic organisms (see Section 6, Aquatic toxicity and EC-ECB, 2004) with invertebrates and/or algae being the most sensitive organism groups. The available data on algae indicate a chronic toxicity potential with NOErC being 0.067 mg/L for methylcyclohexane and 0.94 mg/L for cyclohexane, resulting in chronic classification for both substances.

- Toxicological profile:

Methylcyclohexane and cyclohexane show similar toxicokinetic behaviour (see Section 7.1, Toxicokinetics). For those toxicological endpoints, for which both substances have been tested, similar levels of toxicity have been observed. Thus, both substances are of low acute toxicity via the oral and inhalation route, but fulfil the classification criteria for Aspiration toxicity (Category 1) and Narcotic effects (STOT-SE 3) according to Regulation (EC) No 1272/2008. Both substances were not skin and eye irritating in experimental studies, but are classified for Skin irritation (Category 2), mainly based on their defatting properties. Methylcyclohexane and cyclohexane have been extensively studied for repeated dose toxicity mainly via inhalation. For methylcyclohexane, the lowest chronic NOAEC for systemic effects is 400 ppm (1600 mg/m³) in rats, while for cyclohexane the most reliable subchronic systemic NOAEC in rats and mice is 2000 ppm (6880 mg/m³). Applying a time-extrapolation factor of 3 (ECHA, 2010), the chronic NOAEC for cyclohexane would be 667 ppm (2293 g/m³), which would be in the same order of magnitude as for methylcyclohexane. Both substances have been tested negative for mutagenicity in vitro.

Conclusion:

In order to avoid the need to test methylcyclohexane for every endpoint for which information gaps are identified, the read-across approach is applied for the assessment of environmental and human health hazards. Thus, environmental and human health effects are predicted where applicable from adequate and reliable data for cyclohexane by interpolation to methylcyclohexane in accordance with Annex XI, Item 1.5 of Regulation (EC) No 1907/2006.

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

References

Breitmaier, E. and Jung, G. (2005). Organische Chemie. Grundlagen , Stoffklassen, Reaktionen, Konzepte, Molekülstruktur. 5th ed. Georg Thieme Verlag, Stuttgart.

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/

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.


Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro genetic toxicity studies were negative.

Short description of key information:
Gene mutation in bacteria: negative Ames test with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A, with and without metabolic activation (OECD Guideline 471, GLP).
Cytogenicity: negative results in in vitro mammalian chromosome aberration test in Chinese hamster lung (CHL/IU) cells, with and without metabolic activation (OECD Guideline 473, GLP).
Gene mutation in mammalian cells: negative results in mammalian cell gene mutation assay (mouse lymphoma L5178Y cells), with and without metabolic activation (similar to OECD Guideline 476, read-across from cyclohexane).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on substance specific studies and read-across from the structurally similar reference substance cyclohexane, the available data on in vitro genotoxicity of methylcyclohexane do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.