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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Ames test): Negative based on read-across from alpha-Terpineol which was tested in an OECD TG 471


Gene mutation in mammalian cells: Negative based on read across from alpha-Terpineol, which was tested in an MLA according to OECD TG 476


Chromosome aberration in mammalian cells (human lymphocytes): Negative based on read-across from Terpineol multi, which was tested according to OECD TG 473.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The Terpene hydrocarbon alcohols and their genetic toxicity is based on read-across from alpha-Terpineol and Terpineol multi. The executive summary of the source information on the substance is presented below, followed by the read-across rationale.


Alpha-Terpineol and the Ames test


For alpha-Terpineol an Ames test equivalent to OECD 471 is available (Reliability 2). In this study, S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were exposed to test concentrations between 1 µg and 1000 µg/plate in the presence and absence of mammalian metabolic activation system liver S9 homogenate, from male Sprague-Dawley rats and Syrian golden hamsters injected with Aroclor 1254 at 500 mg/kg body weight. The test substance was tested for mutagenicity at different dose concentrations with both direct plate incorporation and pre-incubation methodology. Alpha-Terpineol caused no dose-related response in the number of histidine auxotroph revertants. The positive controls induced the appropriate responses in the corresponding strains. Based on these results alpha-Terpineol was concluded to be not mutagenic in the reverse mutation assay in bacteria.  


Alpha-Terpineol and its effects in the MLA


For alpha-Terpineol, a mammalian cell gene mutation assay is available which was conducted similarly to OECD guideline 476 (Reliability 2). Mouse lymphoma L5178Y cells cultured in vitro were exposed to test concentrations between 0.14 µg/mL and 0.65 µg/mL in the presence and absence of metabolic activation with liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats.  Alpha-Terpineol was tested for cytotoxicity at concentrations up to an upper limit of 10000 µg/plate and the doses selected for testing were within the range yielding approximately 0-90 % cytotoxicity. In both non-activated and S9-activated conditions, response was negative at a dose 0.14-0.65 µg/mL. The positive controls ethylmethylsulfonate (without metabolic activation) and 3-methylcholanthrene (with metabolic activation) induced the appropriate response. Under conditions of the test, alpha-Terpineol was concluded to be not mutagenic in mammalian cells.


Terpineol multi and its Chromosome aberrations in mammalian cells


For Terpineol Multi, an in vitro chromosome aberration test according to OECD guideline 473 and in compliance with GLP is available (Reliability 1) in which, human primary lymphocyte cultures were exposed to Terpineol Multi in DMSO at concentration range of 5.598-1543 μg/mL, for 3 + 17 h (treatment + recovery) with metabolic activation (2% S-9 fraction of Aroclor 1254-induced male Sprague-Dawley rats), and for 3 + 17 h or 20 + 0 h (treatment + recovery) without metabolic activation for a preliminary cytotoxicity test. In the main test, two experiments were performed at concentrations up to 600 µg/mL without S-9 and up to 800 µg/mL with S-9 and the following concentrations were selected for analysis: Experiment 1: Without S-9 (treatment: 3 h): 0, 350, 425 and 450 μg/mL; with S-9 (treatment: 3 h): 0, 300, 550 and 625 μg/mL. Experiment 2: Without S-9 (treatment: 20 h): 0, 75, 200 and 225 μg/mL; with S-9 (treatment: 3 h): 0, 400, 550, 625 and 650 μg/mL. Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control ranges. Positive controls (4-nitroquinoline-N-oxide at 2.5 and 5 µg/mL without S-9 and cyclophosphamide at 10, 20 and 30 µg/mL with S-9) induced the appropriate response. Treatment of cells with Terpineol Multi in the presence or absence of S-9 in both experiments resulted in frequencies of cells with structural or numerical aberrations that were generally similar to those observed in concurrent vehicle controls for all concentrations analysed. Numbers of aberrant cells (excluding gaps) in treated cultures fell within the normal range with the exception of one culture at the highest concentration analysed with S-9 in experiment 1 (625.0 µg/mL). However, the aberration frequency (excluding gaps) in the replicate culture at 625.0 µg/mL in experiment 1 and in all other cultures analysed in experiments 1 and 2 fell within the normal range. Under the test conditions, Terpineol-Multi is not considered as clastogenic in human lymphocytes.  


Genotoxic properties of Terpene hydrocarbon alcohols using read across from alpha-Terpineol (CAS# 98-55-5) and Terpineol Multi (CAS# 8000-41-7).


 


Introduction and hypothesis for the analogue approach


Terpene hydrocarbon alcohols have the following constituent types of substances: Solely hydrocarbons-terpene type, Alcohol-type, Ketone-type and Ether-type all having a saturated or unsaturated cyclic hydrocarbon backbone.For this substance no genotoxicity information is available. In accordance with Article 13 of REACH, lacking information can be generated by other means than experimental testing, i.e. applying alternative methods such as QSARs, grouping and read-across. For assessing the genotoxicity, information from the constituent Terpineol is used, which can represent the Terpene hydrocarbon alcohols.


Hypothesis: Terpene hydrocarbon alcohols have the same genotoxicity as alpha-Terpineol or Terpineol multi.


Available information: Alpha –Terpineol was negative in an Ames test (OECD TG 471, Rel. 2, non-GLP). This substance was also negative in the genemutations in mammalian cells (MLA, OECD TG 476, Rel. 2, non-GLP). Terpineol multi was negative for in vitro chromosome aberrations (OECD TG 473, Rel. 1)


Target chemical and source chemical(s)


Constituent types of the target substance and chemical structure of the source substances are shown in the data matrix, including physico-chemical properties and toxicological information, thought relevant for genotoxicity.


Purity / Impurities


Constituent types of the target substance are covered by the presented constituent types, there are no other constituent that impacts the genotoxicity.


Analogue approach justification


According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is documented below.


Analogue selection: For Terpene hydrocarbon alcohols their key constituents alpha-Terpineol and Terpineol Multi, a multi-constituent substance with alpha-Terpineol as its main constituent, were selected as source because for genotoxicity information is available, which can be used for read across.


Structural similarities and differences: The Terpene hydrocarbon alcohol constituent types have alpha-Terpineol/Terpineol as key constituents and therefor the same structures. The differences with the other constituent types can be presented as follows. Alcohol-type has a saturated or unsaturated cyclohexyl ring with a secondary or tertiary alcohol. The ones with the saturated bonds are expected to be less electrophilic compared to the ones with the unsaturated bones. The unsaturated ones can also represent the Solely hydrocarbon-terpene type. The latter type has a very similar backbone but often have an additional unsaturated bond in the ring instead of an alcohol group. Also the Ketones have similar rings but the functional group is a ketone instead of an alcohol. These groups, alcohol, double bond, ketone have similar electrophilicity. The Ether in the aromatic Ether is not very electrophilic either. Its double bond adjacent to the aromatic ring is more electrophilic. In view of its presence in the Terpene hydrocarbon alcohols < 4%, this reactivity is not expected to have an impact on the overall genotoxicity profile.


Toxico-kinetic: All Terpene hydrocarbon alcohol constituents have molecular weights, are liquids and physico-chemical properties, log Kow, that present potential absorption via all routes.


Metabolism: Terpene hydrocarbon alcohols metabolites result in similar metabolites as those from alpha-Terpineol and Terpineol Multi. The first metabolic step will result in primary, secondary or tertiary alcohols if these are not already constituents as such.


Genotoxic reactivity: The Terpene hydrocarbon constituents have similar electrophilicity and therefore similar reactivity compared to alpha Terpineol and Terpineol multi. This absence is also indicated by Adams et al. (2011) and Belsito et al. (2008). The saturated Alcohol and Ketones types are slightly less and cis-Anethole is slightly more reactive. These have lower presence in the substance Terpene hydrocarbon alcohols and there the reactivity of this substance can be presented with alpha-Terpineol and Terpineol multi.


Uncertainty of the prediction: There are no uncertainties other than those already addressed above.


Data matrix


The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data Matrix.


Conclusions on genetic toxicity for hazard and risk assessment


For Terpene hydrocarbon alcohols no genotoxicity information as such is available but for its key constituent alpha-Terpineol or Terpineol multi such information is present and can be used for read across to fill this gap. When using read across the result derived should be applicable for C&L and/or risk assessment, cover an exposure period duration comparable or longer than the corresponding method and be presented with adequate and reliable documentation. This documentation is presented in the current text. Alpha-Terpineol is negative for genemutations in bacterial and mammalian cell (OECD TG 471 and 476, Rel. 2, non-GLP). Terpineol multi is negative for chromosomal aberrations in vitro (OECD TG 471, Rel. 1). This information can be used for read across to Terpene hydrocarbon alcohols.


Final conclusion: Terpene hydrocarbon alcohols are negative in genemutations in bacterial and mammalian cells and for chromosomal aberrations.


Data matrix supporting the genotoxicity read across to the Terpene hydrocarbon alcohols from alpha-Terpineol and Terpineol multi.






























































































































Terpene hydrocarbon alcohols



Terpineol hydrocarbon alcohols



Terpineol alpha and multi



 



Target



Source



Structure



Not applicable



 


(α-Terpineol and γ-Terpineol)



CAS



Not applicable



98-55-5 and 8000-41-7



EC No.



945-149-0



202-680-6 /232-268-1



Reach registration



2018



Registered



Molecular weight



136-154



154



Phys-chem properties



 



 



Appearance



Liquid



Liquid



Log Kow



3.3-5.5; IFF



2.6



Identity, Constituent type (%)



100%



 



Solely hydrocarbons



 



 



Limonene type



0-15



 



Alcohol type



 



>80



Tertiary alcohols



40-90



 



Secondary alcohols



7-40



 



Ketone type



-



 



Camphor-Type



0-17%



 



Ether type



 



 



Aromatic ether type



<4%



 



Human health-Genotoxicity



 



 



Genemutation in bacterial cells



Negative


(Read across)



Negative


 (OECD TG 471, alpha-Terpineol)



Genemutation in mammalian cells



Negative


(Read across)



Negative


(OECD TG 471, alpha-Terpineol)



Chromosomal aberrations



Negative


(Read across)



Negative


(OECTG 474, Terpineol multi)



 


References:


Adams et al. The FEMA GRAS assessment of aliphatic and aromatic terpene hydrocarbons


used as flavor ingredients. Food and Chemical Toxicology 49 (2011) 2471-94.


 


Belsito et al. A toxicologic and dermatologic assessment of cyclic and non-cyclic terpene alcohols when used as fragrance ingredients. Food and Chemical Toxicology. 46 (2008a) S1-71.

Justification for classification or non-classification

Based on the negative result for genemutations in bacterial and mammalian cells and negative in the chromosomal aberration assay the substance does not need to be classified for genotoxicity according to EU CLP (EC No. 1272/2008 and its amendments).