Registration Dossier

Administrative data

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: Negetive 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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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: Read-across information
Justification for type of information:
The information is based on read across.The read-across justification is presented in the Endpoint summary Genetic toxicity. The accompanying files are also attached there.
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: read-across from alpha-Terpineol
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay based on read across.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
non-GLP
Justification for type of information:
This information is used for read-across to Terpene hydrocarbon alcohols
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no details about test substance and solvent or vehicle control
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 prepared from male Sprague-Dawley rats and Syrian golden hamsters injected with Aroclor 1254 at 500 mg/kg body weight
Test concentrations with justification for top dose:
Ranged between 10 µg/plate and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation) and preincubation


DURATION
- Preincubation period: 30 min
- Exposure duration: 48 h
Evaluation criteria:
Test article had to induce doubling the mean number of revertants / plate
Statistics:
No details given in study report
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Alpha-Terpineol was not mutagenic in the Ames test in both plate incorporation and preincubation methods, and with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria conducted similarly to OECD guideline 471, TA98, TA100, TA1535, TA1537 and TA1538 strains of S. typhimurium  were exposed to Alpha-terpineol at concentrations between 1µg and 1000 µg/plate in the presence and absence of mammalian metabolic activation system liver S9 homogenate, from male Sprague-Dawley rats and Syriyan golden hamsters injected with Aroclor 1254 at 500 mg/kg body weight.

Alpha-terpineol was tested for mutagenicity at different dose concentrations with both direct plate incorporation and preincubation 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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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: Read-across information
Justification for type of information:
The information is based on read across. The read-across justification is presented in the Endpoint summary Genetic toxicity. The accompanying files are also attached there.
Reason / purpose:
read-across source
Vehicle / solvent:
-
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: all strains/cell types tested / read-across from Terpineol multi
Conclusions:
The substance is not clastogenic in human lymphocytes, based on read across.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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
Justification for type of information:
This information is used for read-across to Terpene hydrocarbon alcohols
Qualifier:
according to
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
Remarks:
without metabolic activation Migrated to IUCLID6: 2.5 and 5 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 10, 20 and 30 µg/mL
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
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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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

Conclusions:
Under the test conditions, Terpineol-Multi is not considered as clastogenic in human lymphocytes.
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
Remarks:
Type of genotoxicity: gene mutation
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: Read-across information
Justification for type of information:
The information is based on read across. The read-across justification is presented in the Endpoint summary Genetic toxicity. The accompanying files are also attached there.
Reason / purpose:
read-across source
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
Remarks on result:
other: read-across from alpha-Terpineol
Conclusions:
The substance is not mutagenic in an in vitro mammalian cell gene mutation assay with mouse lymphoma L5178Y cells.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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
Remarks:
no details about test substance and individual results.
Justification for type of information:
This information is used for read-across to Terpene hydrocarbon alcohols
Qualifier:
equivalent or similar to
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:
In a mammalian cell gene mutation assay with mouse lymphoma L5178Y cells cultured in vitro, the substance gave negative reults both in the presence and absence of metabolic activation (liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats).
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 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 followingconstituent 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 substanceare 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, wereselected 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 alcoholsare negative in genemutations in bacterial and mammalian cells and for chromosomal aberrations.

Data matrix supporting the genotoxicity read acrossto theTerpene 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

4.9 (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).