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EC number: 238-620-0 | CAS number: 14576-08-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
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- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test with Orange Flower Ether (OECDTG471): negative
Genemutation in mammalian cells (OECDTG 476) with Terpineol-alpha as an analogue for Orange Flower Ether): negative
Chromosomal aberration assay (OECDTG 473 with Terpineol-multi as an analogue for Orange Flower Ether): negative.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 9 June 2011 - 22 July 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- METI, MHLW and MAFF
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May 2008
- Qualifier:
- according to guideline
- Guideline:
- other: EPA (TSCA) OPPTS harmonised guidelines
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- - S. typhimurium: his-locus
- E. coli: trp-locus - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbitone/ß-Naphthoflavone induced rat liver S9-mix
- Test concentrations with justification for top dose:
- - Preliminary toxicity test (TA 100, WP2 uvrA, with and without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
- Experiment 1 (all strains, with and without S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate
- Experiment 2 (TA 98, TA 100, TA 1535, TA 1537, with S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
- Experiment 2 (WP2 uvrA, with S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
- Experiment 2 (all strains, without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- Experiment 1:
METHOD OF APPLICATION: in agar (plate incorporation);
DURATION: Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3
Experiment 2:
METHOD OF APPLICATION: preincubation
DURATION: Preincubation period: 20 min; Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
RANGE-FINDING/SCREENING STUDIES:
The test item was toxic at 5000 μg/plate to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.
ADDITIONAL INFORMATION ON CYTOTOXICITY
In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 1500 μg/plate in the absence and presence of S9-mix. In the main test (pre-incubation method) the test item induced a much greater toxic response with weakened bacterial background lawns initially noted from 15 and 150 μg/plate in the absence and presence of S9-mix respectively. The sensitivity of the tester strains to the toxicity of the test item varied between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to either the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on experimental methodology. - Conclusions:
- The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and the E.coly assay performed according to OECD guideline 471.
- Executive summary:
In a GLP compliant study, in accordance with OECD guideline 471, the test substance was examined for its possible mutagenic activity using a the bacterial reverse mutation test. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 15 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using doses ranging between 0.15 and 5000 μg/plate. The sensitivity of the assay and the efficacy of the S9-mix were validated with negative and positive control plates. In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 1500 μg/plate in the absence and presence of S9-mix. In the main test (pre-incubation method) the test item induced a much greater toxic response with weakened bacterial background lawns initially noted from 15 and 150 μg/plate in the absence and presence of S9-mix respectively. The sensitivity of the tester strains to the toxicity of the test item varied between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to either the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The test item was therefore considered to be non-mutagenic under the conditions of the test.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From April 04 to June 23, 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: cultures prepared from the pooled blood of three male donors
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- 2% S9 fraction of Aroclor 1254-induced male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- Range-finder experiment: 5.598-1543 μg/mL (with and without S-9)
Main study:
- Experiment 1: Without S-9: 0, 350, 425 and 450 μg/mL; with S-9: 0, 300, 550 and 625 μg/mL
- Experiment 2: Without S-9: 0, 75, 200 and 225 μg/mL; with S-9: 0, 400, 550, 625 and 650 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- PREPARATION OF CULTURES: Whole blood cultures pooled from three healthy, non-smoking male volunteers were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 µg/mL gentamycin, so that the final volume following addition of S9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1 °C for approximately 48 hours and rocked continuously.
METHOD OF APPLICATION: In medium
DURATION
- Exposure duration: 3 or 20 hours, 37 ± 1 ºC
- Fixation time (start of exposure up to harvest of cells): 20 or 20.75 hours
SPINDLE INHIBITOR (cytogenetic assays): Colchicine, 1 µg/mL for 2 hours
STAIN (for cytogenetic assays): Giemsa (4% v/v)
NUMBER OF REPLICATIONS: Duplicates
NUMBER OF CELLS EVALUATED: At least 1000 cells/dose were counted in cytotoxicity test to determine the mitotic index; at least 200 metaphase cells/dose were analysed for chromosomal aberrations
DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index
OTHER EXAMINATIONS:
- Cells with structural aberrations including or excluding gaps, polyploidy, hyperdiploidy or endoreduplication were recorded during the study. - Evaluation criteria:
- For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range were observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p ≤ 0.05)
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
- Test article was considered as positive in this assay if all of the above criteria were met.
- Test article was considered as negative in this assay if none of the above criteria were met.
- Results which only partially satisfied the above criteria were dealt with on a case by case basis. - Statistics:
- - Statistical method used was Fisher's exact test.
- Proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
- Probability values of p ≤ 0.05 were accepted as significant. - Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see Table 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolality: No marked changes in osmolality or pH were observed at the highest concentration tested in the range-finding cytotoxicity experiment (1543 µg/mL, equivalent to 10 mM), compared to the concurrent vehicle controls.
- Solubility/Precipitation: Miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 174.6 mg/mL. Solubility limit in culture medium was approximately 873.1-1746 µg/mL as indicated by precipitation at the higher concentration which persisted for approximately 20 hours after test article addition.
RANGE-FINDING/SCREENING STUDIES:
- In the range-finding cytotoxicity study, precipitation was observed at or above 200 μg/mL and complete cytotoxicity was seen at or above 925.8 µg/mL tested with or without S-9.
- See table 2 for more data
COMPARISON WITH HISTORICAL CONTROL DATA: Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control (normal) ranges. - Conclusions:
- Under the test conditions, Terpineol-Multi is not considered as clastogenic in human lymphocytes under the conditions of this test
- Executive summary:
In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, 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 (Lloyd 2010). 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.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- no data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- no details about test substance and individual results
- GLP compliance:
- no
- Type of assay:
- other: In vitro mammalian cell gene mutation test
- Target gene:
- no data
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Fisher's media, Gibco, Grand Island, NY
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver S9 from Aroclor 1254-induced male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- Between 0.14 µg/mL and 0.65 µg/mL
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- for the test with out metabolic activation
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Remarks:
- for the test with metabolic activation
- Details on test system and experimental conditions:
- Toxicity test: Cells at a concentration of 6 10^5/mL were exposed for 4 h to a range of concentrations from 0.0005 to 10000 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1 °C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. For each assay, there were 2-4 solvent controls.
Mutagenicity assay: A total of 1.2 10^7 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 °C, washed twice with growth medium, and maintained at 37 ± 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 10^5/mL at 24 h intervals. They were then cloned (1 10^6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. Plates were incubated at 37 ± 1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter or ProtoCol colony counter. Only colonies larger than 0.2 mm in diameter were counted. The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from 0.2 to 1.1 mm. - Evaluation criteria:
- Doubling of the mutant frequency over the cocurrent solvent treated control value. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
- Statistics:
- No details given in study report
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The doses of chemical selected for testing were within the range yielding approximately 0-90 % cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Test results confirmed previous results showing negative response both with and without metabolic activation.
- Executive summary:
In a mammalian cell gene mutation assay conducted similarly to OECD guideline 476, mouse lymphoma L5178Y cells cultured in vitro were exposed to alpha-terpineol at 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 cytotoxic concentration up to an upper limit of 10000 µg/plate. In both nonactivated 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.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Reliability is 2 because of the use of read across
- Justification for type of information:
- Executive summary: Orange Flower Ether, using read across from Terpineol alpha, is negative in the chromosomal aberration assay. The full read across is presented in the Endpoint summary and attached in the present study record. It includes also the MLA endpoint.
Orange Flower Ether has similar genotoxicity as Terpineol-multi and Terpineol Alpha. Orange Flower Ether has a methyl ether as the functional group while Terpineol-multi and Terpineol Alpha have an alcohol group, which is not expected to influence the genotoxicity.
Structural similarities and differences: The backbone of Orange Flower Ether (target) and the Terpineols is the same, it contains of a cyclohexyl ring with one double bond and a methyl-group attached to the head-position. The difference between target and source is the substitution on the para-position: Orange Flower Ether has a methyl ether and both Terpineols an alcohol. This difference is not expected to influence the genotoxicity of these substances.
Toxico-kinetic similarities and differences: Absorption: The source substances and target substances have similar absorption potential based on the similarity in chemical structure and physico-chemical properties. Though the target is an ether and the source substances are alcohols resulting in slight differences, the physico-chemical values are still expected to result in full absorption via the oral, dermal and inhalation route. All three substances are liquids. The vapour pressures of the Orange flower ether and source Terpineol Alpha are between 1 and 10 Pa, it is somewhat higher for Terpineol-multi, which vapour pressure is high likely because of a volatile impurity or constituent Metabolism: Orange Flower Ether metabolises into Terpineol Alpha because of the de-methylation of the ether bond. Thereafter the metabolic pathway is anticipated to be alike.
Toxico-dynamic aspect: Reactivity: Both substances are expected to have equal limited reactivity the methyl ether nor the alcohol functional group is a reactive group for mutagenicity. Therefore reactivity of the source and target chemical is expected to be similar.
Uncertainty of the prediction: There is no remaining uncertainty, in view of similarities in structure of the parent substances and Orange Flower Ether metabolizing into Terpineol Alpha read across is justified. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: Reference to Lloyd 2010
Referenceopen allclose all
Table 2: Range-finder experiment: mitotic index determinations
Treatment |
Mitotic index (%) |
||||||||
(µg/mL) |
3+17 hours, -S-9 |
3+17 hours, +S-9 |
20+0 hours, -S-9 |
||||||
|
A |
B |
MIH* |
A |
B |
MIH* |
A |
B |
MIH* |
Vehicle |
5.7 |
7.4 |
- |
7.0 |
5.1 |
- |
5.9 |
6.7 |
- |
5.598 |
6.2 |
NT |
5 |
6.7 |
NT |
0 |
6.0 |
NT |
5 |
9.330 |
5.6 |
NT |
15 |
5.7 |
NT |
6 |
6.3 |
NT |
0 |
15.55 |
7.7 |
NT |
0 |
5.1 |
NT |
16 |
5.4 |
NT |
14 |
25.92 |
4.5 |
NT |
31 |
6.2 |
NT |
0 |
5.3 |
NT |
16 |
43.19 |
6.0 |
NT |
8 |
7.8 |
NT |
0 |
6.4 |
NT |
0 |
71.99 |
6.2 |
NT |
5 |
6.2 |
NT |
0 |
6.0 |
NT |
5 |
120.0 |
6.8 |
NT |
0 |
7.1 |
NT |
0 |
4.9 |
NT |
22 |
200.0 |
6.4 |
NT |
2P |
4.0 |
NT |
34P |
4.5 |
NT |
29P |
333.3 |
6.1 |
NT |
7P |
4.8 |
NT |
21P |
2.2 |
NT |
65P |
555.5 |
0.0 |
NT |
100P |
4.0 |
NT |
34P |
0.0 |
NT |
100P |
925.8 |
T |
NT |
100P |
T |
NT |
100P |
T |
NT |
100P |
1543 |
T |
NT |
100P |
T |
NT |
100P |
T |
NT |
100P |
NT = Not tested; P = Precipitation observed at treatment; T = Toxic
*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%
(where T = treatment and C = negative control)
Table 3: Results summary
Treatment |
Concentration (mg/mL) |
Cytotoxicity (%) |
% Cells with Chromosome Aberrations (Excluding Gaps) |
Historical (%)# |
Statistical significance |
Experiment 1 |
|||||
3+17.75 hour -S-9 |
Vehiclea |
- |
0.50 |
0-3 |
- |
|
350.0 |
0 |
0.50 |
|
NC |
|
425.0 |
29 |
1.00 |
|
NC |
|
450.0 |
50 |
0.50 |
|
NC |
|
*NQO, 2.50 |
ND |
8.00 |
|
p ≤ 0.001 |
3+17.75 hour +S-9 |
Vehiclea |
- |
0.50 |
0-3 |
- |
|
300.0 |
0 |
1.00 |
|
NC |
|
550.0 |
35 |
3.00 |
|
NC |
|
625.0 |
50 |
5.00 |
|
NC |
|
*CPA, 10.00 |
ND |
25.83 |
|
p ≤ 0.001 |
Experiment 2 |
|||||
20+0 hour -S-9 |
Vehiclea |
- |
1.00 |
0-3 |
- |
|
75.00 |
18 |
0.00 |
|
NC |
|
200.0 |
34 |
1.50 |
|
NC |
|
225.0 |
53 |
1.00 |
|
NC |
|
*NQO, 5.00 |
ND |
29.37 |
|
p ≤ 0.001 |
3+17 hour +S-9 |
Vehiclea |
- |
0.50 |
0-3 |
- |
|
400.0 |
7 |
0.50 |
|
NC |
|
550.0 |
34 |
1.50 |
|
NC |
|
625.0 |
39 |
2.50 |
|
NC |
|
650.0 |
50 |
1.00 |
|
NC |
|
*CPA, 20.00 |
ND |
42.11 |
|
p ≤ 0.001 |
a Vehicle control was DMSO
* Positive control
#95th percentile of the observed range
NC = Not calculated
ND = Not determined
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
For genotoxicity information of three in vitro tests are available: the Ames test with Orange Flower Ether, a genemutation in mammalian cells with Terpineol-alpha and a cytogenicity test (CAB) with the analogue Terpineol-multi of which the summaries are presented below. Thereafter the read across justification is presented.
Ames test
In a GLP compliant study, in accordance with OECD guideline 471, the test substance was examined for its possible mutagenic activity using a the bacterial reverse mutation test. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 15 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using doses ranging between 0.15 and 5000 μg/plate. The sensitivity of the assay and the efficacy of the S9-mix were validated with negative and positive control plates. In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 1500 μg/plate in the absence and presence of S9-mix. In the main test (pre-incubation method) the test item induced a much greater toxic response with weakened bacterial background lawns initially noted from 15 and 150 μg/plate in the absence and presence of S9-mix respectively. The sensitivity of the tester strains to the toxicity of the test item varied between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to either the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The test item was therefore considered to be non-mutagenic under the conditions of the test.
There are no other genetic toxicity in vitro studies available for our target substance. Therefore information available for two different structural analogues are used the complete the assessment.
For alpha-Terpineol an in vitro gene mutation test with mammalian cells is available, and an in vitro chromosomal aberration test was performed with Terpineol multi (a multi-constituent substance with alpha-Terpineol as its main constituent and gamma-Terpineol as the minor constituent). In the toxico-kinetic section) the constituents of alpha-Terpineol and Terpineol multi are presented. These terpineols are expected to have a similar genotoxicity profile because of their similarity in structure. It can also be seen that all constituents of Terpineol multi have a similar backbone, which is the cyclohexyl ring. The attached methyl groups are para-positioned. There are two functional groups. The first one is the tertiary alcohol, which is not reactive because no additional reactive groups are adjacent to this alcohol. The second functional group is the double bond, at the para–position but can be inside or outside the cyclohexyl ring. These differences are thought to be of minor importance for the genotoxicity potential
MLA study
In a mammalian cell gene mutation assay conducted similarly to OECD guideline 476, mouse lymphoma L5178Y cells cultured in vitro were exposed to alpha-Terpineol at 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 (Seifried 2006). Alpha-Terpineol was tested for cytotoxic concentration up to an upper limit of 10000 µg/plate. 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.
Chromosome aberration study
In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, 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 (Lloyd 2010). 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.
Genotoxicity of Orange Flower Ether (CAS #14576-08-0) using read across from substances Terpineol-multi (CAS #8000-41-7) and Terpineol Alpha (CAS #98-55-5).
Introduction and hypothesis for the analogue approach
Orange Flower Ether is an ether attached to a cyclohexyl ring with one double bond with a methyl-group attached at the para-position. For this substance a genotoxicity test in bacterial strains is available but no experimental information on cytogenicity or gene mutation is mammalian cells. In accordance with Article 13 of REACH, lacking information can be generated by mean other than experimental tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the genotoxicity of Orange Flower Ether the analogue approach is selected because for two closely related analogues, Terpineol-multi, containing mostly Terpineol Alpha, and Terpineol Alpha, a reliable chromosomal aberration test and gene mutation study in human cells are available.
Hypothesis: Orange Flower Ether has similar genotoxicity as Terpineol-multi and Terpineol Alpha. Orange Flower Ether has a methyl ether as the functional group while Terpineol-multi and Terpineol Alpha have an alcohol group, which is not expected to influence the genotoxicity.
Available information: The target and source substances are negative in well conducted genotoxicty tests in bacterial strains, receiving reliabilities of 1 (OECD TG 471). The source chemical Terpineol-multi is negative in the in vitro cytogenicity assay (OECD TG 473). The source chemical Terpineol Alpha is negative in the mouse lymphoma assay (OECD TG 476). The genotoxicity tests are all performed according to OECD guidelines and well conducted and therefore receive reliabilities of 1 when under GLP or 2 when GLP is unknown.
Target chemical and source chemical(s)
Chemical structures of the target chemical and the source chemicals are shown in the data matrix, including physico-chemical properties and toxicological information, thought relevant for repeated dose of both substances. Two registration dossiers were generated for Terpineol-multi being a multi-constituent (but containing mainly Terpineol-alpha) and Terpineol-alpha (being a mono-constituent. Because of the presence of these two REACH dossiers and experimental testing done with both type of analogues the reference is made to these dossiers separately. The key reference substance is Terpineol-alpha or also presented as Terpineols.
Purity / Impurities
Orange Flower Ether is a mono-constituent and contains impurities with the functional group, the ether bond, is absent or at another spot in the structure. This is similar to Terpineol-multi in which the functional group, the alcohol can be absent or at a different spot in the structure. These impurities and/or minor constituents are not affecting the genotoxic profile. Therefore it is not expected that the impurities / minor constituents of the source and target chemicals affect the read-across justification.
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 with a common functional group and/or metabolism result in the same metabolite. 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.
In accordance with ECHA (2015, RAAF) Terpineol-multi and Terpineol Alpha were selected as being the closest analogues with information that can cover the endpoints for Orange Flower Ether.
Structural similarities and differences:The backbone ofOrange Flower Ether (target) and the Terpineols is the same, it contains of a cyclohexyl ring with one double bond and a methyl-group attached to the head-position. The difference between target and source is the substitution on the para-position: Orange Flower Ether has a methyl ether and both Terpineols an alcohol. This difference is not expected to influence the genotoxicity of these substances.
Toxico-kinetic similarities and differences:Absorption:The source substances and target substances have similar absorption potential based on the similarity in chemical structure and physico-chemical properties. Though the target is an ether and the source substances are alcohols resulting in slight differences, the physico-chemical values are still expected to result in full absorption via the oral, dermal and inhalation route. All three substances are liquids. The vapour pressures of the Orange flower ether and source Terpineol Alpha are between 1 and 10 Pa, it is somewhat higher for Terpineol-multi, which vapour pressure is high likely because of a volatile impurity or constituentMetabolism:Orange Flower Ether metabolises into Terpineol Alpha because of the de-methylation of the ether bond. Thereafter the metabolic pathway is anticipated to be alike.
Toxico-dynamic aspect: Reactivity: Both substances are expected to have equal limited reactivity the methyl ether nor the alcohol functional group is a reactive group for mutagenicity. Therefore reactivity of the source and target chemical is expected to be similar.
Uncertainty of the prediction:There is no remaining uncertainty, in view of similarities in structure of the parent substances and Orange Flower Ether metabolizing into Terpineol Alpha read across is justified.In accordance with ECHA guidance (2015, RAAF) the read across would receive a score of 5.
Data matrix
The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data matrix.
Conclusions per endpoint for Hazard assessment and C&L.
For Orange Flower Ether eas well as both source substances (Terpineol-multi and Terpineol-alpha), well-conducted genotoxicity tests in bacterial strains are available, which are clearly negative (OECD TG 471).For the source substance Terpineol-multi a well-conducted mammalian in vitro cytogenicity assay is available (OECD TG 476, Reliability 1) showing absence of cytogenicity. For the source substance Terpineol-alpha a well conducted negative gene mutation in mammalian cells is available (OECD TG 476). Furthermore, no small colonies were found in this MLA test indicating that Terpineol-alpha does not induce chromosomal aberrations.
Final conclusion on hazard, C&L and risk characterisation
Orange Flower Ether is considered negative for genotoxicity based on its negative Ames test and the cytogenicity and gene-mutation information from its analogues. Based on these results the substance does not need to be classified for genotoxicity according to CLP Regulation (EC) No. 1272/2008 and its updates.In view of the absence of genotoxicity a risk characterization for this endpoint is not needed.
Data matrix for the read across to Orange Flower Ether from Terpineol-multi and Terpineol Alpha
Common names |
Orange Flower Ether |
Terpineol-multi |
Terpineol Alpha |
Chemical structures |
|||
CAS no |
14576-08-0 |
8000-41-7 |
98-55-5 |
EC no |
Registration in 2018 (238-620-0) |
232-268-1 |
202-680-6 |
Empirical formula |
C11H20O |
C10H18O |
C10H18O |
Smiles |
O(C([C@@H]1CCC(=CC1)C)(C)C)C |
C([C@@H]1CCC(C)=CC1)(C)(C)O |
C([C@@H]1CCC(C)=CC1)(C)(C)O |
Physico-chemical data |
|
|
|
Molecular weight |
168.28 |
154.25 |
154.25 |
Physical state |
Liquid |
Liquid |
Liquid |
Melting point, °C |
<-20 |
< -20 |
<-20 |
Boiling point, °C |
222.2 |
214 - 225 |
218.9 |
Vapour pressure, Pa |
9.91 at 23°C |
300 at 20°C |
6.48 at 23°C |
Water solubility, mg/L |
85 at 23°C |
2540 at 20°C |
2870 at 23°C |
Log Kow |
4.5 at 25°C |
2.6 at 30°C |
2.6 at 30°C |
Human health endpoints |
|
|
|
Genotoxicity – Ames test |
Negative (OECD TG 471) |
Negative (OECD TG 471) |
Negative (OECD TG 471) |
In vitro cytogenicity |
Read across from Terpineol |
Negative (OECD TG 473) |
Read across from Terpineol-multi |
Genotoxicity - in vitro MLA |
Read across from Terpineol Alpha |
Read across from Terpineol Alpha |
Negative (OECD TG 476) |
Justification for classification or non-classification
Based on the negative results of the Ames test, the mouse lymphoma assay with alpha-Terpineol and the negative result of the in vitro chromosome aberration assay with Terpineol multi (a multi-constituent substance with alpha-Terpineol as its main constituent and gamma-Terpineol as the minor constituent) Orange Flower ether does not need to be classified for genotoxicity in vitro according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its updates.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.