4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

Endpoint summary

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

Reference

Reference 1

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.

Reference 2

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.

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 MI_T/mean MI_C)] 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	Vehicle^a	-	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	Vehicle^a	-	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	Vehicle^a	-	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	Vehicle^a	-	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

^#95^th percentile of the observed range

NC = Not calculated

ND = Not determined

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.

Reference 3

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.

Reference 4

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

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.