2,6-dimethyloctan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: Mutagenicity in bacteria with Tetrahydromyrcenol (OECD TG 471): negative

Chromosomal aberrations: In vitro cytogenicity : negative (read-across from Dihydromyrcenol tested in OECD TG 473)

Mouse lymphoma assay (MLA) In vitro gene mutation in mammalian cells: negative (read-across from Dihydromyrcenol tested in (OECD TG 476)

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:

30 Jan 2001 - 16 Feb 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix

Test concentrations with justification for top dose:

- 5 to 5000 µg/plate in the presence of S9
- 1.5 to 5000 µg/plate in the absence of S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: according to guidelines

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard plate incorporation

DURATION: Exposure duration 48-72h

NUMBER OF REPLICATIONS: The experiment was performed in triplicate, and repeated in full after an interval of at least 3 days.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn measurement and reduction in revertant colonies compared to the controls

Statistics:

Estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level was done, using a X2-test (Mohn and Ellenberger, 1977).

Key result

Species / strain:

S. typhimurium, other: TA1537 and TA102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA1535 and TA100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA100 and TA102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed

HISTORICAL CONTROL DATA:
A historical overview of the revertant frequencies of the strains used in the Freiburger Labor fur Mutagenitätsprüfung of the year 2000:
Mix: -S9 +S9
Strain: spontaneous solvent control spontaneous solvent control
TA1535: 19±5 (12/31) 19±5 (11/31) 13±5 (7/23) 14±7 (6/36)
TA1537: 10±4 (5/15) 11±4 (8/16) 15±3 (11/20) 15±3 (11/21)
TA98: 25±8 (13/41) 22±7 (15/36) 26±6 (17/36) 24±5 (14/30)
TA100: 114±19 (84/142) 105±15 (76/129) 115±18 (90/149) 108±16 (85/145)
TA102: 299±34 (225/351) 281±33 (233/332) 336±51 (261/420) 322±41 (267/388)

The historical data of positive controls of the year 2000 are given in the form: strain, mutagen (concentration of mutagen in μg/plate), mean of revertants per plate (minimum/maximum). The data of test without S9 are: TA1535, NaN3 (0.7), 631±268 (180/1065); TA1537, 9-AA (50), 259±81 (131/362); TA98, 2-NF (2.5), 361±126 (190/632); TA100, NaN3 ( 0.7), 434183 (329/665); TA102, Mitomycin C (0.15) 811±156 (577/1061). The data of the tests with lot KH3900 of S9 are: TA1535, 2-AA (0.8), 161±50 (103/264); TA1537, 2-AA (1.7), 223±66 (143/331); TA98, 2-AA (0.8), 532±159 (211/845); TA100, 2-AA (0.8), 675±216 (352/1156); TA102, 2-AA (0.8), 584±174 (383/897). Additional data of the tests with lot KH3900 of S9 are: TA98, B(a)P (5.0), 523±30 (504/558); TA100, B(a)P (5.0), 801±58 (746/862).

The number of spontaneous revertants observed using each of the five strains was close to those previously established in the laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979). The results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the fall activity of the metabolizing system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
-In the absence of S9-mix TETRAHYDROMYRCENOL was bacteriotoxic towards the strains TA1537 and TA102 at 500 µg/plate, towards the strains TA1535 and TA100 at 1500 µg/plate, and towards the strain TA98
at 5000 µg/plate.
-In the presence of S9-mix TETRAHYDROMYRCENOL was bacteriotoxic towards the strain TA1537 at 500 µg/plate, towards the strains TA100 and TA102 at 1500 µg/plate and towards the strain TA98 at 5000 μg/plate.

Conclusions:

Under the conditions of this study, TetrahydroMyrcenol was determined to be not mutagenic.

Executive summary:

The mutagenic activity of TetrahydroMyrcenol was evaluated in accordance with OECD TG 471 and according to GLP principles. The test was performed as a standard plate incorporation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and background lawn, was observed. In the absence of S9-mix test material was toxic towards the strains TA1537 and TA102 at 500µg/plate, towards the strains TA1535 and TA100 at 1500µg/plate, and towards the strain TA98 at 5000µg/plate. In the presence of S9-mix test material was bacteriotoxic towards the strain TA1537 at 500µg/plate, towards the strains TA100 and TA102 at 1500µg/plate and towards the strain TA98 at 5000 μg/plate. No precipitation was observed at any of the concentrations. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 TA102), both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 November 2009 to 28 January 2010 (Experimental Phase)

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

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: Lymphocytes obtained from the peripheral circulation of human volunteers

Details on mammalian cell type (if applicable):

The cell-cycle time for lympohcytes from donors used in this study was determined using bromodeoxyuridine (BrdU) incorporation to measure the number of first, second and third division metaphase cells and to calculate an average generation time (AGT). The AGT for donors from this laboratory has been approximately 17 hours under typical exposure conditions.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 6.10, 12.19, 24.38, 48.75, 97.5, 195, 390, 780 and 1560 microg/ml

Main Experiment, 4-Hour Exposures with or without S9, followed by 20 hour treatment free: 0, 100, 200, 300, 400, 600 and 800 microg/ml.

Main Experiment, without S9, 24-Hours: 0, 12.5, 25, 50, 100, 200 and 300 microg/ml

Vehicle / solvent:

dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (without S9); Cyclophosphamide (with S9)

Details on test system and experimental conditions:

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): ) 4-hour exposure followed by 20-hour culture in treatment-free media prior to cell harvest

STAIN (for cytogenetic assays): 5% Gurrs Giemsa

NUMBER OF CELLS EVALUATED: 2000 lymphocyte cell nuclei

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Harvested and fixed Lymphocytes were re-suspended in several ml of fresh fixative before centrifugation and re-suspended in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Slides when dried were stained with 5% Gurrs Giemsa for 5 minutes, rinsed, dried and cover slips applied using mounting medium

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): The first 100 consecutive well-spread metaphases from each culture were counted

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: When possible, the first 100 consecutive well-spread metaphases from each culture were counted. If there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated with 50 cells. If a cell had 44-48 chromosomes, any gaps, breaks or rearrangements were recorded according to the simplified system of Savage (1976) as recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence (%) reported.

DETERMINATION OF CYTOTOXICITY
Preliminary Toxicity Test:
A preliminary toxicity test was performed with or withut metabolic activation using a 4-hour exposure time followed by a 20-hour recovery period and a continuous exposure for 24 hours without metabolic activation. After qualitative microscopic analysis of culture slides from each treatment, appropriate dose levels were selected for mitotic index evaluation. Mitotic index was used to assess material toxicity and to select dose levels for the main test.

- OTHER:
Cell Cultures:Cells were grown in Eagle's Minimal Essential Medium (MEM) with HEPES buffer, supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal calf serum. Cells were maintained at 37 deg C in a 5% CO2 atmosphere. Lymphocytes from freshly heparinized whole blood were stimulated to divide with the addition of phytohemagglutinin (PHA) at approximately 90 microg/ml.
Duplicate lympohcyte cultures (A and B) were used for each dose level and were prepared by mixing the following components and dispersing into sterile plastic flasks: 9.05 ml MEM, 10% (FCS); 0.1 ml Li-heparin; 0.1 ml phytohemagglutinin; and 0.75 ml heparinised whole blood.

Test and Control Materials:
The test material was dissolved in dimethyl sulfoxide (DMSO) at a maximum concentration of 1560 microg/ml, providing a final maximum concentration of approximately 10 mM. There were no significant pH changes in the media and the osmolality did not increase by more than 50 mOsm. The test material was applied to the test system within 2 hours of preparation and the stability of the formulation was assumed for this duration.
Mitomycin C (Sigma Batch No. 104K0332) dissolved in Minimal Essential Medium was used at 0.4 and 0.2 microg/ml in the 4/20-hour and 24-hour exposure groups without S9, respectively. Cyclophosphamide (Acros batch No. A0164185) dissolved in DMSO was used at 5 microg/ml.

Treatment With S9:
After approximately 48 hours of incubation, cell cultures were transferred to tubes and centrifuged. Approximately 9 ml of culture medium was removed, reserved and replaced with the required volume of MEM (including serum) and 0.1 ml of either vehicle control or test material was added to each tube. The positive control received 0.1 ml of the appropriate solution. Aliquots of 1 ml of 20% S9 mixture was added to cultures in the Preliminary Toxicity Test and in the main experiment.
After 4 hours at 37 deg C, cultures were centrifuged, the medium removed and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation, the wash medium was removed and replaced with the original culture medium and incubation continued a further 20 hours.

Treatment Without S9:
After approximately 48 hours of incubation, cell cultures were transferred to tubes and centrifuged. Approximately 9 ml of culture medium was removed, reserved and replaced with the required volume of MEM (including serum) and 0.1 ml of either vehicle control, test material or positive control solution was added to each tube giving total final volumes of 10 ml. After 4 hours at 37 deg C, cultures were centrifuged and test medium removed and replaced with an 8 ml wash of MEM. After further centrifugation, the wash medium was removed and replaced with the original culture medium and the cells returned to the incubator for a further 20 hours.
In the 24-hour continuous exposure group without S9 the culture medium was incubated at 37 deg C for 24 hours.

Main Experiment:
The main experiment was conducted as follows: i) 4-hour exposure without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest; ii) 4-hour exposure with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest; iii) 24-hour continuous exposure to the test material without S9-mix prior to cell harvest.

Cell Harvest:
Mitosis was arrested with addition of demecolcine (Colcemid, 0.1 microg/ml) two hours before the required harvest time. After incubation, cells were centrifuged, the cell medium discarded and cells resuspended in 0.075 M hypotonic KCl. After approximately 14 minutes (including centrifugation), most of the solution was removed and discarded. Cells were resuspended and fixed by dropping the KCl suspension into methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least 3 times and cells stored at approximately 4 deg C for at least 4 hours to ensure complete fixation.

Metaphase Spreads:
Lymphocytes were re-suspended in several ml of fresh fixative before centrifugation and re-suspended in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Slides when dried were stained with 5% Gurrs Giemsa for 5 minutes, rinsed, dried and cover slips applied using mounting mediumMETHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

Evaluation criteria:

Qualitative Slide Assessment:

Slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.

Mitotic Index:

A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of vehicle control.

Scoring of Chromosomal Damage:

When possible, the first 100 consecutive well-spread metaphases from each culture were counted. If there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated with 50 cells. If a cell had 44-48 chromosomes, any gaps, breaks or rearrangements were recorded according to the simplified system of Savage (1976) as recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Cells with 69 chromosomes or more were scored as polyploid cells and the incidence (%) reported.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fischer's Exact test.

Key result

Species / strain:

other: human lymphocyte cultures

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:

In a preliminary toxicity test concentrations of 6.10 to 1560 microg/ml (10 mM) were employed. A greasy/oily precipitate of the test material was present in parallel blood-free cultures at the end of exposure at and above 390 microg/ml in the 4/20-hour and 24-hour continuous exposure groups without S9. In the 4/20-hour exposure group with S9 a greasy/oily precipitate of the test material was present at and above 780 microg/ml. Hemolysis was seen at and above 97.5 microg/ml at the end of exposure in the 4/20-hour and 24-hour exposure groups in the absence of S9 and at and above 48.75 micorg/ml in the 4/20-hour exposure group with S9. Microscopic analysis of slides prepared from the exposed cultures indicated metaphase cells were present up to 390 microg/ml in the 4/20-hour exposures with and without S9. In the 24-hour cultures, the maximum dose at which metaphases were present was 195 microg/ml.

MAIN EXPERIMENT:

The ratios of numbers of total aberrations/cell for the 4/20-hour exposures without metabolic activation (S9) for the control, 100, 200 and 300 microg/ml concentrations were: (with gaps) 2/200, 3/200, 2/200 and 3/200; (without gaps) 1/200, 0/200, 1/200 and 1/200.

The ratios of numbers of total aberrations/cell for the 4/20-hour exposures with metabolic activation (S9) for the control, 200, 300 and 400 microg/ml concentrations were: (with gaps) 5/200, 2/200, 1/200 and 2/200; (without gaps) 3/200, 2/200, 1/200 and 1/200.

The ratios of numbers of total aberrations/cell for the 24-hour continuous exposures without metabolic activation (S9) for the control, 25, 50, 100 and 200 microg/ml concentrations were: (with gaps) 4/200, 7/200, 6/200, 4/200 and 2/200; (without gaps) 0/200, 6/200, 2/200, 2/200 and 1/200.

The test material did not induce statistically significant increases in the numbers of polyploid cells at any dose level in any exposure group.

In a parallel Mouse Lymphoma Assay using L5178Y cells (Harlan Laboratories Project No. 2197/0057), which was capable of detecting a clastogenic response, there was no evidence of a toxicologically significant response with this same test material. This was taken as scientific justification to confirm that a repeat of the exposure group with metabolic activation was not required.

COMPARISON WITH HISTORICAL CONTROL DATA:

Control culture aberration frequencies were acceptable and were within historical control ranges for in-house data (data not shown).

Dihydromyrcenol did not induce any statistically significant increases in the frequencies of cells with aberrations, under any of the exposure conditions, using dose ranges that included a dose level that induced near optimum mitotic inhibition in the 4/20 -hour exposure group with S9 and in the 24 -hour exposure group. Optimum toxicity was not achieved in the 4/20 -hour exposure group without S9 due to the steepness of the toxicity curve. All vehicle control and positive control materials produced frequencies of cells with aberrations with expected ranges based on in-house historical control data.

Conclusions:

2,6-dimethyloct-7-en-2-ol (dihydromyrcenol) did not induce a statistically significant increase in the frequency of cells with chromosomal aberrations either with or without rat liver S9 metabolic activation. The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Dihydromyrcenol was assessed for its ability to induce chromosomal aberrations in cultured human lymphocytes In Vitro. Cells were exposed to the test substance in 4-Hour Exposures with or without S9, followed by 20 hour treatment free. Doses used with S9: 0, 100, 200, 300, 400, 600 and 800 µg/ml, without S9, 24-Hours: 0, 12.5, 25, 50, 100, 200 and 300 µg/ml. The sensitivity of the test system and the activity of the metabolic activation were demonstrated by using the direct acting mutagen Mitomycin C and the promutagen Cyclophosphamide as positive controls. Both substances increased significantly the rate of structural chromosome aberrations.

Exposure of cultured human lymphocytes to Dihydromyrcenol with and without metabolic activation did not result in statistically significant increases of the rate of structural chromosome aberrations. Dihydromyrcenol is therefore considered negative in the chromosome aberration test under the conditions of these assays.

Reference 3

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: information is derived from analogue

Justification for type of information:

The information for Tetrahydromyrcenol is retrieved from Dihydromyrcenol. The read across justification is presented in the Genetic toxicity Endpoint summary and the accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: human lymphocyte cultures

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Tetrahydromyrcenol is considered negative for chromosomal aberrations based on read across from Dihydromyrcenol.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 November 2009 to 07 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

No. 440/2008 of 30 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase heterozygous system, TK +/- to TK -/- conversion

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK +/- 3.7.2c mouse lymphoma cell line, obtained from Dr. J. Cole of the MRC Cell Mutation Unit, University of Sussex, Brighton, UK

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test:
0, 6.09, 12.19, 24.38, 48.75, 97.5, 195, 390, 780, 1560 micrograms/mL

Main Experiment, 4-Hour, Without S9:
0, 12.5, 25, 50, 100, 125, 150, 175, 200 micrograms/mL

Main Experiment, 4-Hour, With S9:
0, 25, 50, 100, 125, 150, 175, 200, 250 micrograms/mL

Main Experiment, 24-Hours, Without S9:
0, 6.25, 12.5, 25, 50, 75, 100, 125, 150 micrograms/mL

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without S9 activation Migrated to IUCLID6: Sigma batch 1419706 15108051 at 400 micrograms/mL (4 hour) and 150 micrograms/mL (24-hour)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 activation Migrated to IUCLID6: Acros batch A0164185 at 2 micrograms/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension;
- Cell density at seeding (if applicable): 4 hour exposures: 1 x 10^6 cells/ml in 10 ml aliquots, 24 hours exposures: .3 x 10^6 cells/ml in 10 ml

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): expression period of two days
- Selection time (if incubation with a selection agent): ten to fourteen days of incubation
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 4 μg/ml 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. the MTT vital stain was taken up by viable cells to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

NUMBER OF CELLS EVALUATED:The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

- OTHER:
Cell Culture
Cells were stored in liquid nitrogen at -196°C. Cells were cultured at 37°C with 5% CO2 in air in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media). Cells had a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) were used during the course of the study. Master cultures of stock cells were checked and found to be mycoplasma free.

Preparation of test and control materials
The test material was dissolved in dimethyl sulfoxide (DMSO) and the appropriate dilutions were made. The maximum dose level investigated in the preliminary toxicity test was 1560 μg/ml which was equivalent to approximately 10 mM.. Analysis for concentration, homogeneity and stability of the test material preparations were not a requirement of the test method and were therefore not performed.
Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) at 400 μg/ml and 150 μg/ml for the 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) 2 μg/ml was used as the positive control in the presence of metabolic activation.

Preliminary toxicity test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/ml, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 6.09 to 1560 μg/ml for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10^5 cells/ml.
The cultures were incubated and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/ml. After a further 24 hours, the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth values (SG). The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth Value (%RSG).
Results from the preliminary toxicity test were used to set the test material dose levels for the mutagenicity experiment. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/ml or 10 mM.
ii) The presence of excessive precipitate where no test material-induced toxicity was observed.
iii) Test material-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).

Mutagenicity test (main test)
4-Hour exposures, with and without metabolic activation
An exponentially growing stock culture of cells was set up to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test material (12.5 to 200 μg/ml in the absence of metabolic activation, and 25 to 250 μg/ml in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to bring the total volume to 20 ml.
The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

24-Hour exposures, without metabolic activation
Cells were counted and processed (as above) to give 0.3 x 10^6 cells/ml in 10 ml duplicate cultures established in 25 cm2 tissue culture flasks. To each culture was added 2 ml of the treatment dilutions (0.2 ml for the positive control) and sufficient R10 medium to give a final volume of 20 ml. The dose range of the test material was 6.25 to 150 μg/ml. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker for 24 hours.
Cell processing following treatment periods
At the end of the treatment periods for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/ml. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/ml.
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies were scored manually by eye using qualitative judgment. Large colonies were defined as those covering approximately ¼ to ¾ of the surface of the well and were generally no more than one or two cells thick. As a rule of thumb, all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies were normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. the MTT vital stain was taken up by viable cells to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 10^-6 for the TK+/- locus in L5178Y cells at this testing laboratory. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10^-6 mutant frequency per survivor are not normally acceptable and will be repeated. Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. A Global Evaluation Factor (GEF) value was set following International Workshop (Moore et al., 2003; Moore et al, 2006) at 126 x 10^-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgment will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Robinson W D et al (1989) Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp102-140.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The maximum dose level used was limited by test material toxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test:The results for Relative Suspension Growth (%RSG) are as shown in Table 1:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A precipitate of the test material was observed at and above 195 μg/ml in the 4-hour exposure group in the absence of metabolic activation, and at and above 390 μg/ml in both the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation. The precipitate was observed to become greasy and oily in appearance at 1560 μg/ml in both of the 4-hour exposure groups.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutagenicity test results
4-Hour exposures with and without metabolic activation (Table 2):
There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any significant reductions in (%V) viability in either the absence or presence of metabolic activation, therefore indicating that no residual toxicity had occurred. Near optimum levels of toxicity were achieved in the absence of metabolic activation. Optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation due to the sharp onset of toxicity, despite using a very narrow dose interval. However, a dose level that exceeded the usual acceptable upper limit of toxicity was plated for viability and TFT resistance for each of the exposure groups. It was therefore considered that with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including the dose levels that exceeded the usual upper limit of acceptable toxicity, or in the 24-hour exposure group where optimum levels of toxicity were achieved, the test material had been adequately tested. The excessive toxicity observed at 200 μg/ml in the absence of metabolic activation, and at 250 μg/ml in the presence of metabolic activation, resulted in these doses not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

24-Hour exposure without metabolic activation (Table 3):
As was seen in the Preliminary Toxicity Test, there was evidence of a marked reduction in % RSG and RTG values in cultures dosed with the test material. There was also evidence of a modest reduction in (%V) viability, therefore indicating that residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved. The positive control induced acceptable levels of toxicity.
The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had an effect on the toxicity of the test material. The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.
The test material induced a small statistically significant dose related (linear-trend) in the mutant frequency x 10^-6 per viable cell (Table 9). However, statistically significant increases in mutant frequency were not observed at any of the individual dose levels, the GEF was not exceeded at any of the individual dose levels, and the mutant frequency values observed were within the acceptable range for vehicle controls. Therefore, the response was considered to be spurious and of no toxicological significance. Precipitate of the test material was not observed at any of the dose levels.

Remarks on result:

other: all strains/cell types tested

Table 1: Preliminary Toxicity Test Results

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	%RSG (+ S9) 4-Hour Exposure	%RSG (-S9) 24-Hour Exposure
0	100	100	100
6.09	93	103	94
12.19	87	116	81
24.38	87	111	80
48.75	90	103	62
97.5	84	101	34
195	0	28	0
390	0	0	0
780	0	0	0
1560	0	0	0

Table 2: Experimental Results

4-Hour Test Without S9
Treatment (mg/mL)	%RSG	RTG	MF
0	100	1.00	118.34
12.5*	95
25	92	1.16	91.83
50	88	1.05	101.08
100	84	0.99	104.88
125	73	0.81	120.90
150	37	0.38	122.92
175**	1	0.03	143.07
200***	0	-	-
Linear trend			NS
EMS
400	74	0.60	684.79
4-Hour Test With S9
0	100	1.00	149.96
12.5*	92
25	90	0.98	91.37
50	83	0.88	147.57
100	83	0.97	150.12
125	77	0.81	106.33
150	50	0.50	116.61
175**	9	0.12	111.16
200*	0	-	-
Linear trend			NS
CP
2	48	0.24	781.04

* Not plated for viability or 5-TFT resistence.

** Treatment excluded from statistics due to toxicity.

Table 3: Experimental Results

24-Hour Test Without S9
Treatment (mg/mL)	%RSG	RTG	MF
0	100	1.00	115.95
6.25*	104
12.5	108	0.92	91.35
25	99	0.96	98.55
50	71	0.66	121.85
75	46	0.43	101.71
100	30	0.16	149.79
125	11	0.10	158.82
150*	2	-	-
Linear trend			p < 0.05
EMS
150	66	0.45	1007.15

* Not plated for viability or 5-TFT resistence.

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was not considered to be mutagenic under the conditions of the test. Therefore, Dihydromyrcenol does not need to be classified as mutagenic according to Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

The objective of this in vitro assay (OECD TG 476) was to evaluate the ability of Dihydromyrcenol to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line, using Trifluorothymidine (TFT) as selection agent. The test material was dissolved in Dimethylsulfoxide (DMSO). A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/ml, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 6.09 to 1560 μg/ml for all three of the exposure groups. The test material produced dose-related toxicity, starting at 195 ug/ml with and without activation. A precipitate of the test material was observed at and above 195 μg/ml in the 4-hour exposure group in the absence of metabolic activation, and at and above 390 μg/ml in both the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation.

 

The 4-hour exposure to dihydromyrcenol, with and without metabolic activation was performed in duplicate (A + B), at eight dose levels of the test material (12.5 to 200 μg/ml in the absence of metabolic activation, and 25 to 250 μg/ml in the presence of metabolic activation), vehicle and positive controls. For the 24-hour exposure without metabolic activation, the dose range of the test material was 6.25 to 150 μg/ml. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker.

 

At 4 hours of exposure, there was evidence of a marked reduction in % RSG and RTG values in cultures dosed with the test material. There was also evidence of a modest reduction in (%V) viability, therefore indicating that residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved. The positive control induced acceptable levels of toxicity. The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had an effect on the toxicity of the test material. The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

 

Dihydromyrcenol was evaluated as negative with and without metabolic activation in the L5178Y mouse lymphoma mutation assay under the conditions used in this assay. It was concluded that Dihydromyrcenol does not need to be classified as mutagenic according to Annex I of the CLP Regulation (1272/2008/EC).

Reference 5

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: Information is derived from analogue

Justification for type of information:

For Tetrahydromyrcenol the in vitro genemutation in mammalian cell were assessed by using read across from Dihydromyrcenol. The read across justification is presented in the Genetic toxicity Endpoint summary. The accompanying files are also attached there.

Reason / purpose for cross-reference:

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 maximum dose level used was limited by test material toxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was not considered to be mutagenic under the conditions of the test. Therefore, Dihydromyrcenol does not need to be classified as mutagenic according to Annex I of the CLP Regulation (1272/2008/EC). This result was used for read-across to TetrahydroMyrcenol.

Executive summary:

The objective of this in vitro assay (OECD TG 476) was to evaluate the ability of Dihydromyrcenol to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line, using Trifluorothymidine (TFT) as selection agent. The test material was dissolved in Dimethylsulfoxide (DMSO). A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/ml, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 6.09 to 1560 μg/ml for all three of the exposure groups. The test material produced dose-related toxicity, starting at 195 ug/ml with and without activation. A precipitate of the test material was observed at and above 195 μg/ml in the 4-hour exposure group in the absence of metabolic activation, and at and above 390 μg/ml in both the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation.

 

The 4-hour exposure to dihydromyrcenol, with and without metabolic activation was performed in duplicate (A + B), at eight dose levels of the test material (12.5 to 200 μg/ml in the absence of metabolic activation, and 25 to 250 μg/ml in the presence of metabolic activation), vehicle and positive controls. For the 24-hour exposure without metabolic activation, the dose range of the test material was 6.25 to 150 μg/ml. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker.

 

At 4 hours of exposure, there was evidence of a marked reduction in % RSG and RTG values in cultures dosed with the test material. There was also evidence of a modest reduction in (%V) viability, therefore indicating that residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved. The positive control induced acceptable levels of toxicity. The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had an effect on the toxicity of the test material. The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

 

Dihydromyrcenol was evaluated as negative with and without metabolic activation in the L5178Y mouse lymphoma mutation assay under the conditions used in this assay. It was concluded that Dihydromyrcenol does not need to be classified as mutagenic according to Annex I of the CLP Regulation (1272/2008/EC). This result was used for read-across to TetrahydroMyrcenol.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For Tetrahydromyrcenol an Ames test is available. The mammalian genotoxicity information for Tetrahydromyrcenol is derived from read across using Dihydromyrcenol as an analogue. First the executive summary of the experimental studies of Dihydromyrcenol are presented and thereafter the read across justification. The accompanying files are also attached here.

Tetrahydromyrcenol: Ames (Gene mutation in bacteria)

They key study is an Ames study for TetrahydroMyrcenol and was performed in accordance with OECD 471 guideline and according to GLP principles. The test was performed as a standard plate incorporation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and background lawn, was observed. In the absence of S9-mix, the test material was bacteriotoxic towards the strains TA1537 and TA102 at 500µg/plate, towards the strains TA1535 and TA100 at 1500µg/plate, and towards the strain TA98 at 5000µg/plate. In the presence of S9-mix test material was toxic towards the strain TA1537 at 500µg/plate, towards the strains TA100 and TA102 at 1500µg/plateand towards the strain TA98 at 5000 μg/plate. No precipitation was observed at any of the concentrations.Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 TA102), both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

Dihydromyrcenol: Chromosomal aberration (in vitro cytogenicity)

The key study is an in vitro chromosome aberration test in cultured human lymphocytes (OECD TG 473). The test was used for read-across to TetrahydroMyrcenol. Cells were exposed to the test substance in 4-Hour Exposures with or without S9, followed by 20 hour treatment free. Doses used with S9: 0, 100, 200, 300, 400, 600 and 800 µg/ml, without S9, 24-Hours: 0, 12.5, 25, 50, 100, 200 and 300 µg/ml. The sensitivity of the test system and the activity of the metabolic activation were demonstrated by using the direct acting mutagen Mitomycin C and the promutagen Cyclophosphamide as positive controls. Both substances increased significantly the rate of structural chromosome aberrations. Exposure of cultured human lymphocytes to Dihydromyrcenol with and without metabolic activation did not result in statistically significant increases of the rate of structural chromosome aberrations. Dihydromyrcenol is therefore considered negative in the chromosome aberration test under the conditions of these assays.

Dihydromyrcenol MLA: in vitro gene mutation in mammalian cells

The key study is an in vitro gene mutation assay with Dihydromyrcenol (OECD TG 476). The study was used for read-across to TetrahydroMyrcenol. The objective was to evaluate the ability of Dihydromyrcenol to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line, using Trifluorothymidine (TFT) as selection agent. The test material was dissolved in Dimethylsulfoxide (DMSO). The 4-hour exposure to Dihydromyrcenol, with and without metabolic activation was performed in duplicate (A + B), at eight dose levels of the test material (12.5 to 200 μg/ml in the absence of metabolic activation, and 25 to 250 μg/ml in the presence of metabolic activation), vehicle and positive controls. For the 24-hour exposure without metabolic activation, the dose range of the test material was 6.25 to 150 μg/ml. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker. At 4 hours of exposure, there was evidence of a marked reduction in % RSG and RTG values in cultures dosed with the test material. There was also evidence of a modest reduction in (%V) viability, therefore indicating that residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved. The positive control induced acceptable levels of toxicity. The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had an effect on the toxicity of the test material. The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily. Dihydromyrcenol was evaluated as negative with and without metabolic activation in the L5178Y mouse lymphoma mutation assay under the conditions used in this assay.

Tetrahydromyrcenol (Cas no18479-57-7)and its non-genotoxicity using read across from Dihydromyrcenol (18479-58-8)

Introduction and hypothesis for the read across

Tetrahydromyrcenol is a branched C8 alkyl chain to which a tertiary alcohol is attached at the end of the alkyl chain.For Tetrahydromyrcenol(target) no gene mutation data in mammalian (mouse lymphoma) cells and no cytogenicity information are available. Therefore, additional information is used in accordance with Article 13 of REACH where it is said that lackinginformation should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, SARs, grouping and read-across.For assessing the genotoxicity in mammalian the analogue approach is selected because for one closely related analogue, Dihydromyrcenol such genotoxicity information is available.

Hypothesis: Tetrahydromyrcenol has the same genotoxicity in mammalian cells as Dihydromyrcenol.

Available information: For Tetrahydromyrcenol a negative Ames study is available (OECD TG 471) but other genotoxicity information is absent. Dihydromyrcenol has been tested in a well conducted MLA and a chromosomal aberration test (OECD Guideline 476 and 473 under GLP, Kl 1). No genotoxicity was found in these tests.

Target and Source chemical(s):

Chemical structures of the Tetrahydromyrcenol (the target) and are shown in the Data matrix including physico-chemical properties and toxicological information, thought relevant for genotoxicity.

Purity / Impurities:

The purity and impurities of the target chemical do not indicate skin sensitisation potential other than indicated by the parent substance. The impurities are all below 10%.

Analogue justification

According REACH Annex XI an analogue approach and structural alert information can be used to replace testing when information from different sources provides sufficient evidence to conclude that this substance has or does not have a particular dangerous property. The result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.

Analogue selection: Dihydromyrcenoland 2,6-Dimethyl-2-heptanol (Cas no. 13254-34-7) wereconsidered as potential analogues with > 80% similarity when using Tanimoto index (MSC). Dihydromyrcenol is considered the more conservative analogue having an electrophilic double bond at one end of the alkyl chain.

Structural similarities and differences:The target Tetrahydromyrcenol and the source chemical Dihydromyrcenol have a 2,6-dimethyl)octan-2-ol backbone and both have a tertiary alcohol functional group at one end of the alkyl chain. The difference is that the alkyl chain of Tetrahydromyrcenol is fully saturated, while Dihydromyrcenol has a double bond at the end of the alkyl chain.

Toxico-kinetics: Beside the structural similarities the molecular weight, the substances being liquids and the physico-chemical properties all indicate a high bioavailability.Metabolism: both substances can be oxidised on their methyl groups and thereafter conjugated. Dihydromyrcenol may in addition be oxidised to an epoxide because of its double bond at one end of the alkyl chain, which result in a higher genotoxic potential for the source compared to the target.

Toxico-dynamic features: Considering reactivity both substances are expected to be similar reactive. As presented above one of the metabolites of Dihydromyrcenol may be more reactive (epoxide formation may occur), which metabolite will not occur in the Tetrahydromyrcenol.

Other experimental information that can support the read across: Tetrahydromyrcenol and Dihydromyrcenol are both negative in the Ames test (OECD TG 471).

Remaining uncertainties:There are no remaining uncertainties because the substances are very alike and if anything Dihydromyrcenol (source) is the more conservative with its potential to generate an epoxide metabolite.

Conclusions per endpoint for hazard and risk assessment

The mammalian in vitro genotoxicity of Tetrahydromyrcenol can be derived from Dihydromyrcenol which is testes for genotoxicity in mammalian cells (MLA, OECD TG 476) and for cytogenicity (OECD TG 473). Dihydromyrcenol is negative in these tests and therefore Tetrahydromyrcenol is negative too.

Final conclusion on hazard: Tetrahydromyrcenol is negative for genotoxicity in mammalian cells. This information is forwarded for the risk assessment.

Data matrix: Information on Tetrahydromyrcenol and its analogue Dihydromyrcenol though relevant to support the genotoxicity read across

Common names	Tetrahydromyrcenol	Dihydromyrcenol
	Target	Source
Chemical structures
CAS no	18479-57-7	18479-58-8
REACH registration	2018	2010
EINECS	242-361-9	242-362-4
Molecular weight	158.29	156.27
Physico-chemical data
Physical state	Liquid	Liquid
Melting point,^oC	<-20	<-20 (ECHA dissemination site)
Boiling point,^oC	201.9	193 (ECHA dissemination site)
Vapour pressure, Pa	9.3	20 (ECHA dissemination site)
Water solubility, mg/l	281.9	939 (ECHA dissemination site)
Log Kow	3.2 (m)	3.25 (ECHA dissemination site)
Human health endpoints
Genotoxicity – Ames test	Not mutagenic (OECD TG 471)	Non mutagenic (OECD TG 471)
Chromosomal aberration	Read across from Dihydromyrcenol	Negative (OECD TG 473)
MLA	Read across from Dihydromyrcenol	Negative (OECD TG 476)

 

Justification for classification or non-classification

Based on the available information Tetrahydromyrcenol does not need to be classified for mutagenicity in accordance with the criteria outline in EU CLP (EC 1272/2008 and its updates).