Melaleuca alternifolia, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

other: A cytogenetic assay in mammalian somatic cells for detection of structural chromosome aberrations using metaphase analysis.

Specific details on test material used for the study:

- Source: Supplied by the sponsor.
- Batch No.: A352
- Appearance: Clear, mobile liquid.
- Expiration date of the batch: 17 December 2011
- Production date of the batch: 17 December 2008
- Storage condition of test material: Room temperature (15-25°C, under nitrogen), in amber glass bottles.
- Formulation: A stock solution of Tea Tree Oil was prepared in dimethyl sulphoxide (DMSO) at a nominal concentration of 25 mg/mL. Appropriate volumes of this stock solution were diluted with Dulbecco's Modified Eagle's (DME) medium to obtain the test concentrations.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Source of cells: ECACC (European Collection of Cells Cultures)
- ECACC Catalogue No.: 86041102
- Lot No.: 05F013
- Expiry date: 15 June 2011
- Suitability of cells: The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology make it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h).
- Checked for mycoplasma infections: Yes, before freezing.
- Details of cell culture and maintenance: Trypsin-EDTA solution is used for cell detachment to subculture. Laboratory cultures were maintained in 75 cm² plastic flasks at 37°C in an incubator with a humidified atmosphere, set at 5% CO2.
- Type and identity of media: V79 cells for use in the study were grown in DME medium, supplemented with L-glutamine (2mM) and 1% of Antibiotic-antimycotic solution and heat-inactivated bovine serum (concentration 10%). During the 3 and 20h treatments with Tea Tree Oil, solvent (negative control) and positive controls, the serum content was reduced to 5% (w/w).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from rat liver. (See 'Any other information on materials and methods' for further details.)

Test concentrations with justification for top dose:

Details of the concentration selection assay are provided in 'Any other information on materials and methods'.

Experiment A with 3/20h treatment/sampling time:
- Without S9 mix: 3.12 μl DMSO/mL (solvent control); 9.76, 19.53, 39.06 and 58.59 μg Tea Tree Oil/mL (metaphase analysis conducted at these concentrations). A treatment at 78.12 μg TTO/mL was not assessed because of very low survival. Positive control (Ethyl methanesulphonate): 1.0 μl/mL.
- With S9 mix (50 μl/mL): 3.12 μl DMSO/mL (solvent control); 9.76, 19.53, 39.06 and 58.59 μg Tea Tree Oil/mL (metaphase analysis conducted at these concentrations). A treatment at 78.12 μg TTO/mL was not assessed because of very low survival. Positive control (N-Nitrosodimethylamine): 1.0 μl/mL.

Experiment B with 20/28h treatment/sampling time:
- Without S9 mix: 2.34 μl DMSO/mL (solvent control); 4.88, 9.76, 19.53 and 39.06 μg Tea Tree Oil/mL (metaphase analysis conducted at these concentrations). Metaphase analysis was not conducted in a treatment at 58.59 μg Tea Tree Oil/mL. Positive control (Ethyl methanesulphonate): 0.4 μl/mL.
Experiment B with 3/28h treatment/sampling time:
- With S9 mix (50 μl/mL): 3.12 μl DMSO/mL (solvent control); 9.76, 19.53, 39.06 and 58.59 μg Tea Tree Oil/mL (metaphase analysis conducted at these concentrations). A treatment al 78.12 μg TTO/mL was not assessed because of very low survival. Positive control (N-Nitrosodimethylamine): 1.0 μl/mL.

Vehicle / solvent:

- Vehicle used: None.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Also N-Nitrosodimethylamine (see 'Any other information on materials and methods').

Details on test system and experimental conditions:

For each experiment, a stock solution of Tea Tree Oil in DMSO was prepared, as described in 'Specific details on test material used for the study' (25 mg/mL). The appropriate amount of stock solution was diluted with medium to obtain the test concentrations. Duplicate cultures were used at each concentration of Tea Tree Oil, along with solvent and the corresponding positive controls for treatment without and with S9 mix. Each culture was seeded with cells (5 x 10^5 cells/dish) in growth medium and after 24h the culture medium of exponentially growing cells was replaced with DME medium containing 5% bovine serum and test substance.

Experiment A: The exposure period was 3h at 37°C. Cells were washed with DME medium at the end of the exposure period and then growth medium was added. Harvesting was performed 20h after the beginning of treatment (approximately 1.5 normal cell cycles).

Experiment B: The exposure period without metabolic activation was 20 h and with metabolic activation was 3h at 37°C. Cells were washed with DME medium at the end of the exposure period and then growth medium was added. Harvesting was done at approximately 2 normal cell cycles (28h) from the beginning of treatment to cover a potential mitotic delay.

Preparation of Chromosomes: Cell cultures were treated with 0.2 μg/mL Colchicine, 2-2.5h prior to harvesting. At the time of harvesting, the cells were swollen with 0.075M KCl hypotonic solution and washed in fixative for ca. 10 min in methanol:acetic acid (3:1, v/v) until the preparation became plasma-free. The cells were subsequently placed onto slides and air-dried. The preparation was stained with 5% Giemsa for subsequent scoring of chromosome aberrations.

Examination of Slides: For control of bias, slides were coded and scored blind. Two hundred metaphase cells containing 22±2 centromeres were evaluated for structural aberrations from each experimental group. Chromatid and chromosome type aberrations (gaps, deletions (breaks) and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells was scored. The nomenclature and classification of chromosome aberrations were reported based upon ISCN (1985) and Savage (1976, 1983).

Rationale for test conditions:

In order to determine the treatment concentrations of Tea Tree Oil to be used in the cytogenetic study, a concentration selection assay (cytotoxicity assay) was performed (see 'Any other information on materials and methods' for further details).

Evaluation criteria:

A test item is regarded as non-clastogenic if: the number of metaphases with structural chromosome aberrations, in all evaluated concentrations, is in the range of the historical control data; and/or no significant increase in the number of metaphases with structural chromosome aberration is observed.
A test item is classified as clastogenic if the following criteria are met: increases in the number of metaphases with aberrant chromosomes are observed, at one or more test concentrations, above the historical control data; the increases are reproducible between replicate cultures and between tests (when the treatment conditions are the same); the increases are statistically significant.

Statistics:

For statistical analysis, Fisher's exact test was utilised. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations (with and without gaps).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and Concentration Selection: Tea Tree Oil was dissolved in DMSO. A clear solution was obtained up to a concentration of 25 mg/mL. At the concentrations analysed for cytotoxicity and chromosome aberrations, no precipitation in the medium was noted.

Concentration selection cytotoxicity: Detailed results of the cytotoxicity assay with Tea Tree Oil are presented in Tables 1 and 2 (see 'Any other information on results'). Following 3 hours treatment, highly toxic effects were observed at concentrations of 78.12 and 156.25 μg/mL with and without metabolic activation. After 20 hours of treatment the cell number was markedly reduced at concentrations of 78.12 and 156.25 μg/mL without metabolic activation. According to these toxicity data, 58.59 μg/mL was chosen as the top concentration for experiment A (3 hours treatment with and without S9 mix) and experiment B (3 hours treatment with S9 mix). A highest concentration of 39.06 μg/mL was selected for experiment B (20 hours of treatment without S9 mix).

Chromosome aberration assay: The cytotoxicity at the highest concentrations was adequate in experiment A and experiment B as indicated by a cell survival reduction of at least 50%.
In Experiment A, Tea Tree Oil did not induce an increase in the number of cells with structural chromosome aberrations without gaps at any examined concentration, either in the absence or presence of metabolic activation, up to and including cytotoxic concentrations.
In Experiment B, a 3 h treatment in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps, thereby confirming the negative results in Experiment A. A similar outcome was obtained without S9 mix using a longer treatment period of 20 h.

No statistical differences between Tea Tree Oil and negative control groups and no dose-response relationships were noted in either experiment.

No polyploid or endoreduplicated metaphases were found after treatment with the different concentrations of Tea Tree Oil.

Validity of the Study:

The historical solvent control data were within the laboratory's normal range for the spontaneous aberration frequency. The positive controls induced increases in aberration frequency, which are signficant. In the control group the percentage of cells with structural aberration(s) without gaps was less than 5%, proving the suitability of the cell line used. The positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and N-Nitrosodimethylamine (1.0 μL/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations. The investigations are therefore considered valid.

Table 1. Summarized results of toxicity and concentration selection assay (3 hours treatment time, with 20 hours sampling time).

Test group	TTO concentration (μg/mL)	S9 mix	Treatment/sampling time (h)	First count x 10^4 /mL	Second count x 10^4 /mL	Mean value x 10^4 /mL	Relative* survival in percent
Untreated control	0	-	3/20	104	102	103	111
Solvent control (6.25 μl DMSO/mL DME)	0	-	3/20	95	91	93	100
Tea Tree Oil	4.88	-	3/20	93	95	94	101
	9.76	-	3/20	92	88	90	97
	19.53	-	3/20	78	86	82	88
	39.06	-	3/20	60	58	59	63
	78.12	-	3/20	10	12	11	12
	156.25	-	3/20	6	4	5	5
Untreated control	0	+	3/20	98	102	100	98
Solvent control   (6.25 μl DMSO/mL DME)	0	+	3/20	104	100	102	100
Tea Tree Oil	4.88	+	3/20	100	102	101	99
	9.76	+	3/20	98	96	97	95
	19.53	+	3/20	78	80	79	77
	39.06	+	3/20	59	63	61	60
	78.12	+	3/20	10	12	11	11
	156.25	+	3/20	5	3	4	4

* Relative to Solvent control

Table 2. Summarized results of toxicity and concentration selection assay (20 and 3 hours treatment times, with 28 hours sampling time)

Test group	TTO concentration (μg/mL)	S9 mix	Treatment/sampling time (h)	First count x 10^4 /mL	Second count x 10^4 /mL	Mean value x10^4 /mL	Relative* survival in percent
Untreated control	0	-	20/28	120	118	119	111
Solvent control (6.25μl DMSO/mL DME)	0	-	20/28	106	108	107	100
Tea Tree Oil	4.88	-	20/28	99	97	98	92
	9.76	-	20/28	83	87	85	79
	19.53	-	20/28	75	71	73	68
	39.06	-	20/28	44	46	45	42
	78.12	-	20/28	2	0	1	1
	156.25	-	20/28	0	0	0	0
Untreated control	0	+	3/28	118	116	117	109
Solvent control (6.25 μl DMSO/mL DME)	0	+	3/28	108	106	107	100
Tea Tree Oil	4.88	+	3/28	109	107	108	101
	9.76	+	3/28	100	104	102	95
	19.53	+	3/28	84	82	83	78
	39.06	+	3/28	64	66	65	61
	78.12	+	3/28	20	16	18	17
	156.25	+	3/28	2	2	2	2

* Relative to Solvent control

Conclusions:

Tea Tree Oil tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in V79 Chinese Hamster lung cells. Therefore, Tea Tree Oil and its metabolite(s) are not considered to be clastogenic in this test system.

Executive summary:

A Chromosome Aberration Assay was carried out according to the OECD Guideline for Testing of Chemicals No. 473.  The test item, Tea Tree Oil (TTO), was tested in V79 cells of the Chinese hamster lung in vitro.  TTO was dissolved in DMSO and a range of test concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation).  In two independent experiments (performed in duplicate) at least 200 well-spread metaphase cells were analysed at the following concentrations and incubation/expression intervals, ranging from low to maximum (< 50% survival) toxicity:

Experiment A with 3/20 h treatment/sampling time:

- Without S9 mix: 9.76, 19.53, 39.06 and 58.59 μg TTO/mL

- With S9 mix: 9.76, 19.53, 39.06 and 58.59 μg TTO/mL

Experiment B with 20/28 h treatment/sampling time:

- Without S9 mix: 4.88, 9.76, 19.53 and 39.06 μg TTO/mL

Experiment B with 3/28 h treatment/sampling time:

- With S9 mix: 9.76, 19.53, 39.06 and 58.59 μg TTO/mL

In Experiment A, there were no increases in the number of cells showing structural chromosome aberrations without gaps, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between TTO and negative control groups and no dose-response relationships were noted.

In Experiment B, the number of cells with structural chromosome aberrations without gaps was not increased when TTO was examined up to cytotoxic concentrations without S9 mix over a prolonged treatment period (20 h). Furthermore, a 3 h treatment with TTO up to cytotoxic concentrations in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps, confirming the negative results in Experiment A. No statistical differences between TTO and negative control groups and no dose-response relationships were noted.

There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.  The validity of the test was demonstrated using Ethyl methanesulphonate (0.4 and 1.0 μL/mL) and N-Nitrosodimethylamine (1.0 μL/mL) as positive controls.

In conclusion, Tea Tree Oil and its metabolite(s) are not considered to be clastogenic in this test system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

- Source: Supplied by the sponsor.
- Lot/batch No. of test material: A352
- Appearance, colour: Liquid, clear-pale straw.
- Expiration date of the lot/batch: 17 December 2011
- Production date of the batch: 17 December 2008
- Storage condition of test material: Room temperature (15-25°C), avoided direct sunlight, nitrogen headspace.

- Formulation: A stock solution of Tea Tree Oil was prepared in dimethyl sulphoxide (DMSO) at a nominal concentration of 50 mg/mL. The stock solution was serially diluted as necessary to prepare appropriate concentrations for treatment.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
- Lot No.: 1661603
- Source of cells: American Type Culture Collection
- Date of receipt: 22 January 2004
- Other: Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/- -mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI 10 medium and incubated at 37 ± 0.5°C in a humidified atmosphere containing approximately 5% CO2 in air. Subcultures were established in an appropriate number of flasks.
- Type and identity of media: Three types of RPMI 1640 medium were prepared. See Table 1 in 'Any other information on materials and methods' for details.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (see Table 2 in 'Any other information on materials and methods' for details)

Test concentrations with justification for top dose:

Details of the concentration selection assay and the concentrations used in the main mutation assays are shown in Table 3 in 'Any other information on materials and methods'.

Vehicle / solvent:

- Solvent used: DMSO. See 'Any other information on materials and methods' for details.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

- Test procedure (main mutation assays): In Assay 1, cells were treated for 3 hours in the presence and absence of S9 mix. In Assay 2, cells were treated for 3 hours in the presence of S9 mix and for 24 hours in the absence of S9 mix. In Assay 3, cells were treated for 24 hours in the absence of S9 mix.

A suitable volume (0.2 mL) of solvent, TTO solutions or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or of 150 mM KCl (treatments without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatment, at least 10^7 cells were placed in each of a series of sterile flasks (culturing surface 75 cm2). For the 24-hour treatment, at least 4x10^6 cells were placed in each of a series of sterile flasks (culturing surface 25 cm2). The treatment medium contained a reduced serum level of 5% (v/v) (RPMI-5).

Duplicate cultures were established for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37°C ± 1°C (approximately 5% CO2 in air). Gentle shaking was used during the 3-hour treatments. After the treatment period, cultures were centrifuged at 2000 rpm for 5 minutes, washed with tissue culture medium and suspended in 20 mL RPMI-10. Viable cells in the individual samples were counted using a haemocytometer. Where sufficient cells survived, cell density was adjusted to a concentration of 2x10^5 cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 25 mL of suspension) or diluted to be plated for survival.

- Plating for survival: Cultures of cell density 2x10^5 cells/mL were further diluted to 8 cells/mL as described in Table 4 (see 'Any other information on materials and methods'). 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates averaging 1.6 cells per well. Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for about two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

- Expression period: To allow expression of the TK- mutation, cultures were maintained in flasks for approximately 3 days. During the expression period, subculturing was performed daily. Cell density was adjusted to a concentration of 2x10^5 cells/mL and transferred to flasks for further growth. On completion of the expression period, at least five concentrations, negative and positive controls and untreated samples were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.

- Plating for viability: At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1x10^4 cells/mL with RPMI-20 in readiness for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL as described in Table 5 (see 'Any other information on materials and methods'). 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates averaging 1.6 cells per well. Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

- Plating for -trifluorothymidine (TFT) resistance: At the end of the expression period, the cell concentration was adjusted to 1x10^4 cells/mL. TFT (300 μg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL. 0.2 mL of each suspension was placed into each well of four, 96-well microplates at 2x10^3 cells per well. Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for approximately two weeks and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test substance.

Evaluation criteria:

The test item is considered to be mutagenic in this assay if all the following criteria are met:
- The assay is valid.
- Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative control values at one or more concentrations.
- The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
- There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
- The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.

Statistics:

Details on the analysis of results and assessment of statistical significance of mutant frequency are provided in 'Any other information on materials and methods'.

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK +/- 3.7.2 C cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Preliminary tests: In the preliminary experiments, cells were exposed to TTO for 3 hours with or without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The highest concentrations selected for the main experiments were expected to result in approximately 10-20% relative survival. In the presence of S9-mix (3 hour treatment), excessive cytotoxicity was observed at 500 μg/mL. The observed cytotoxicity at 250 μg/mL resulted in 16% relative survival, therefore 275 μg/mL was chosen as the highest concentration for the main study. In the absence of S9-mix (3 hour treatment), excessive cytotoxicity was observed at 500 and 250 μg/mL. The observed cytotoxicity at 125 μg/mL resulted in 2.4% relative survival, therefore 120 μg/mL was chosen as the highest concentration for the main study. In the absence of S9-mix (24 hour treatment), excessive cytotoxicity was observed at 500, 250 and 125 μg/mL concentrations. The observed cytotoxicity at 62.5 μg/mL resulted in 25.2% relative survival, therefore 120 μg/mL was chosen as the highest concentration. The concentrations were very closely spaced in the experiments without metabolic activation to obtain the required level of toxicity in the main experiment.

- Mutation assays: In the mutation assays, cells were exposed to TTO for 3 hours with or without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The cells were plated for determination of survival and in parallel subcultured without test item for approximately 3 days to allow expression of the genetic changes. At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

Assay 1: In Assay 1, a 3 hour treatment with metabolic activation and a 3 hour treatment without metabolic activation were performed. Data are presented for survival (Table 6), viability (Table 7) and mutagenicity (Tables 8 and 9). No precipitation of the test item was observed visually in either assay.
In the presence of S9-mix (3 hour treatment), excessive cytotoxicity was observed at 275, 250, 225, 200 and 150 μg/mL concentration. At these concentrations cells were not plated for viability and TFT resistance due to the absence of surviving cells after the expression period. An evaluation was made at concentrations of 100, 75, 50, 25, 10 and 5 μg/mL, although the observed cytotoxicity at 100 μg/mL (relative survival value of 87%) did not met the criteria of 10-20% relative survival value described in the OECD guideline (see Table 6). No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant concentration response to the treatment was indicated by the linear trend analysis (see Table 8).
In the absence of S9-mix (3 hour treatment), excessive cytotoxicity was observed at concentrations of 120, 110, 100 and 90 μg/mL. At these concentrations cells were not plated for survival after treatment or plated for viability and TFT resistance after the expression period due to the low number of surviving cells. Marked cytotoxicity was observed at 80 μg/mL concentration (3% relative survival), which is lower than the acceptance criteria of the OECD guideline (10-20% relative survival). Therefore this concentration was excluded from further evaluation. The observed cytotoxicity at 70 μg/mL resulted in 17% relative survival (see Table 6), which met the acceptance criteria for the highest concentration. Therefore, an evaluation was made at concentrations of 70, 60, 40, 20, 10 and 5 μg/mL. No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant concentration response to the treatment was indicated by the linear trend analysis (see Table 9).

Assay 2: In Assay 2, a 3 hour treatment with metabolic activation and a 24 hour treatment without metabolic activation were performed. Data are presented for survival (Table 10), viability (Table 11) and mutagenicity (Tables 12 and 13). No precipitation of the test item was observed visually in either experiment.
In the presence of S9-mix (3 hour treatment), three concentrations (137.5, 125 and 112.5 μg/mL), additional to the concentration range examined in Assay 1 were included to fulfil the requirements regarding the acceptable level of cytotoxicity at the highest concentration. In this experiment, excessive cytotoxicity was observed at concentrations of 275, 250, 225 and 200 μg/mL. No cells survived on the survival plates and no samples were plated for viability or TFT resistance at these concentrations due to the absence of surviving cells during the expression period. Marked cytotoxicity was observed at 150 and 137.5 μg/mL concentrations (with 1 and 2% relative survival, respectively). Furthermore cells died in these samples during the expression period.
The observed cytotoxicity at 125 μg/mL resulted in 12% relative survival, which met the acceptance criteria of 10-20% of relative survival for the highest concentration (see Table 10). An evaluation was made at concentrations of 125, 112.5, 100, 75, 50, 25, 10 and 5 μg/mL. No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by linear trend analysis (see Table 12).
In the absence of S9-mix (24-hour treatment), excessive cytotoxicity was observed at concentrations of 120, 110, 100, 90, 80, 70 and 60 μg/mL. At these concentrations cells were not plated for survival or maintained through the expression period due to the low number of surviving cells after the treatment. The cytotoxicity at the next concentration of 50 μg/mL resulted in 15% relative survival, which met the acceptance criteria of 10-20% of relative survival for the highest concentration (see Table 10). However, cells in the 50 μg/mL concentration died during the expression period; therefore this sample was not plated for viability or TFT resistance. The cytotoxicity at the next concentration of 40 μg/mL resulted in 46% relative survival (Table 10), which is higher than the acceptance criteria for the highest concentration. An evaluation was made at concentrations of 40, 30, 20, 10 and 5 μg/mL. No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant concentration response to the treatment was indicated by linear trend analysis (see Table 13).

Assay 3: In Assay 1 and Assay 2 of the experiments without metabolic activation, the mutant frequencies of the solvent control samples were below the normal range of the laboratory and lower than the lower limit of the range recommended by the International MLA Workshop. To confirm the negative results and generate more robust data, an additional experiment (Assay 3) was performed. In Assay 3, a 24 hour treatment without metabolic activation was performed. Data are presented for survival (Table 14), viability (Table 15) and mutagenicity (Table 16). No precipitation of the test item was observed visually in this experiment.
In the absence of S9-mix (24 hour treatment), excessive cytotoxicity was observed at concentrations of 120, 110, 100, 90, 80, 70 and 60 μg/mL. At these concentrations cells were not plated for survival or maintained through the expression period due to the low number of surviving cells after the treatment. The observed cytotoxicity at the next concentration of 50 μg/mL resulted in 30% relative survival, which is slightly higher than the acceptance criteria of 10-20% relative survival for the highest concentration (see Table 14). An evaluation was made at concentrations of 50, 45, 40, 30, 20, 10 and 5 μg/mL. No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant concentration response to the treatment was indicated by linear trend analysis (see Table 16).

The Mouse Lymphoma Assay with tea tree oil using L5178Y TK +/- 3.7.2 C cells was considered to be valid for the assessment of potential mutagenicity. Treatment with TTO did not result in a statistically or biologically significant dose-dependent increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9 fraction) in the Mouse Lymphoma Assay.

Table 6. Survival Results - Assay 1.

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells/total number of wells	Plating Efficiency (PE)	Harmonised Relative Survival (%) ^§(%RS)
+	3	A1	275 μg/mL	384/384	0.00000	0
			250 μg/mL	384/384	0.00000	0
			225 μg/mL	384/384	0.00000	0
			200 μg/mL	384/384	0.00000	0
			150 μg/mL	384/384	0.00000	0
			100 μg/mL	52/384	1.24962	87
			75 μg/mL	43/384	1.36840	97
			50 μg/mL	72/384	1.04624	98
			25 μg/mL	70/384	1.06384	102
			10 μg/mL	98/384	0.85355	88
			5 μg/mL	97/384	0.85996	96
			Solvent control	103/384	0.82245	100
			Untreated control	86/384	0.93518	114
			Positive control (CP)	258/384	0.24855	26
-	3	A1	120 μg/mL	ND	ND	ND
			110 μg/mL	ND	ND	ND
			100 μg/mL	380/384	0.00654	0
			90 μg/mL	373/384	0.01817	0
			80 μg/mL	201/384	0.40459	3
			70 μg/mL	114/384	0.75903	17
			60 μg/mL	48/384	1.29965	67
			40 μg/mL	65/384	1.11016	89
			20 μg/mL	75/384	1.02072	92
			10 μg/mL	66/384	1.10062	93
			5 μg/mL	62/384	1.13969	101
			Solvent control	59/384	1.17069	100
			Untreated control	81/384	0.97262	83
			Positive control (NQO)	96/384	0.86643	72

A1 = Assay 1

+ = in the presence of S9-mix

- = in the absence of S9-mix

CP = Cyclophosphamide (4 μg/mL)

NQO: 4-Nitroquinoline-N-oxide (0.15 μg/mL)

Solvent control= 1 % (v/v) DMSO

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed during treatment.

Table 7. Viability Results - Assay 1.

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells / total number of wells	Plating Efficiency (PE)
+	3	A1	275 μg/mL	ND	ND
			250 μg/mL	ND	ND
			225 μg/mL	ND	ND
			200 μg/mL	ND	ND
			150 μg/mL	ND	ND
			100 μg/mL	110/384	0.78135
			75 μg/mL	80/384	0.98038
			50 μg/mL	92/384	0.89303
			25 μg/mL	100/384	0.84092
			10 μg/mL	75/384	1.02072
			5 μg/mL	77/384	1.00427
			Solvent control	89/384	0.91375
			Untreated control	98/384	0.85355
			Positive control (CP)	138/384	0.63962
-	3	A1	120 μg/mL	ND	ND
			110 μg/mL	ND	ND
			100 μg/mL	ND	ND
			90 μg/mL	ND	ND
			80 μg/mL	116/384	0.74816
			70 μg/mL	102/384	0.82854
			60 μg/mL	94/384	0.87959
			40 μg/mL	91/384	0.89986
			20 μg/mL	92/384	0.89303
			10 μg/mL	100/384	0.84092
			5 μg/mL	75/384	1.02072
			Solvent control	75/384	1.02072
			Untreated control	98/384	0.85355
			Positive control (NQO)	74/384	1.02911

A1 = Assay 1

+ = in the presence of S9-mix

- = in the absence of S9-mix

CP = Cyclophosphamide (4 μg/mL)

NQO: 4-Nitroquinoline-N-oxide (0.15 μg/mL)

Solvent control = 1% (v/v) DMSO

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed during the treatment or in the expression period.

Table 8. Mutagenicity Results - Assay 1 (I.).

(3 hour treatment in the presence of S9-mix)

S 9 mix	Treatment period (hours)	Test item or control concentration	Number of empty wells / total number of wells	Number of large colonies / total number of wells	Number of small colonies / total number of wells	Dn^2 /var(Dn) ♦	Mutation frequency
+	3	275 μg/mL	ND	ND	ND	ND	ND
		250 μg/mL	ND	ND	ND	ND	ND
		225 μg/mL	ND	ND	ND	ND	ND
		200 μg/mL	ND	ND	ND	ND	ND
		150 μg/mL	ND	ND	ND	ND	ND
		100 μg/mL	705/768	41/768	22/768	0.057567	54.8
		75 μg/mL	706/768	37/768	25/768	0.271777	42.9
		50 μg/mL	709/768	41/768	18/768	0.149664	44.8
		25 μg/mL	711/768	43/768	14/768	0.095743	45.9
		10 μg/mL	695/768	48/768	25/768	0.013718	48.9
		5 μg/mL	693/768	57/768	18/768	0.000757	51.2
		Untreated control	715/768	34/768	19/768	-	41.9
		Solvent control	700/768	51/768	17/768	-	50.7
		Positive control (CP: 4 μg/mL)	322/768	210/768	236/768	♦♦ 7.33E-11*	679.5*

In linear trend analysis β²/var (β) = 0.03, not significant.

* = Statistically significant compared to the solvent control.

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn²/var(Dn) > 5.24 (at p<0.05).

♦♦ = Evaluated by T-test for independent samples. Significant at p<0.05.

Dn = Difference of log mutant frequency of dose “n” and that of the solvent control

var(Dn) = variance of Dn; β = slope of the curve; var(β) = variance of the slope

Solvent control = 1% (v/v) DMSO

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed in the expression period.

Table 9. Mutagenicity Results - Assay 1 (II.)

(3 hour treatment in the absence of S9-mix)

S9- mix	Treatment period (hours)	Test item or control concentration	Number of empty wells / total number of wells	Number of large colonies / total number of wells	Number of small colonies / total number of wells	Dn^2 /var(Dn) ♦	Mutation frequency
-	3	120 μg/mL	ND	ND	ND	ND	ND
		110 μg/mL	ND	ND	ND	ND	ND
		100 μg/mL	ND	ND	ND	ND	ND
		90 μg/mL	ND	ND	ND	ND	ND
		80 μg/mL	NE	NE	NE	NE	NE
		70 μg/mL	688/768	49/768	31/768	3.959417	66.4
		60 μg/mL	732/768	25/768	11/768	0.268684	27.3
		40 μg/mL	704/768	44/768	20/768	1.040168	48.3
		20 μg/mL	719/768	31/768	18/768	0.060414	36.9
		10 μg/mL	713/768	35/768	20/768	0.552010	44.2
		5 μg/mL	717/768	29/768	22/768	0.000000	33.7
		Untreated control	702/768	41/768	25/768	-	52.6
		Solvent control	717/768	31/768	20/768	-	33.7
		Positive control (NQO: 0.15 μg/mL)	475/768	210/768	83/768	♦♦ 2.96E-9*	233.44*

In linear trend analysis β²/var (β) = 0.33, not significant.

* = Statistically significant compared to the solvent control.

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn²/var(Dn) > 5.24 (at p<0.05).

♦♦ = Evaluated by T-test for independent samples. Significant at p<0.05.

Dn = Difference of log mutant frequency of dose “n” and that of the vehicle control

var(Dn) = variance of Dn; β = slope of the curve; var(β) = variance of the slope

Solvent control = 1% (v/v) DMSO

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed during the treatment or in the expression period.

NE = Not evaluated

Table 10. Survival Results - Assay 2.

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells / total number of wells	Plating Efficiency (PE)	Harmonised Relative Survival (%) ^§ (%RS)
+	3	A2	275 μg/mL	384/384	0.00000	0
			250 μg/mL	384/384	0.00000	0
			225 μg/mL	384/384	0.00000	0
			200 μg/mL	384/384	0.00000	0
			150 μg/mL	379/384	0.00819	1
			137.5 μg/mL	367/384	0.02830	2
			125 μg/mL	278/384	0.20189	12
			112.5 μg/mL	153/384	0.57513	33
			100 μg/mL	73/384	1.03761	73
			75 μg/mL	69/384	1.07284	92
			50 μg/mL	88/384	0.92082	71
			25 μg/mL	117/384	0.74279	63
			10 μg/mL	94/384	0.87959	81
			5 μg/mL	100/384	0.84092	76
			Solvent control	96/384	0.86643	100
			Untreated control	100/384	0.84092	97
			Positive control (CP)	347/384	0.06332	8
-	24	A2	120 μg/mL	ND	ND	ND
			110 μg/mL	ND	ND	ND
			100 μg/mL	ND	ND	ND
			90 μg/mL	ND	ND	ND
			80 μg/mL	ND	ND	ND
			70 μg/mL	ND	ND	ND
			60 μg/mL	ND	ND	ND
			50 μg/mL	64/384	1.11985	15
			40 μg/mL	89/384	0.91375	46
			30 μg/mL	89/384	0.91375	67
			20 μg/mL	76/384	1.01244	86
			10 μg/mL	65/384	1.11016	94
			5 μg/mL	80/384	0.98038	89
			Solvent control	67/384	1.09122	100
			Untreated control	54/384	1.22604	112
			Positive control (NQO)	258/384	0.24855	15

A2 = Assay 2

+ = in the presence of S9-mix

- = in the absence of S9-mix

CP = Cyclophosphamide (4 μg/mL)

NQO: 4-Nitroquinoline-N-oxide (0.1 μg/mL)

Solvent control = 1% (v/v) DMSO

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed during treatment.

Table 11: Viability Results - Assay 2.

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells / total number of wells	Plating Efficiency (PE)
+	3	A2	275 μg/mL	ND	ND
			250 μg/mL	ND	ND
			225 μg/mL	ND	ND
			200 μg/mL	ND	ND
			150 μg/mL	ND	ND
			137.5 μg/mL	ND	ND
			125 μg/mL	91/384	0.89986
			112.5 μg/mL	79/384	0.98825
			100 μg/mL	85/384	0.94249
			75 μg/mL	105/384	0.81043
			50 μg/mL	71/384	1.05498
			25 μg/mL	68/384	1.08196
			10 μg/mL	63/384	1.12969
			5 μg/mL	82/384	0.96495
			Solvent control	81/384	0.97262
			Untreated control	72/384	1.04624
			Positive control (CP)	253/384	0.26078
-	24	A2	120 μg/mL	ND	ND
			110 μg/mL	ND	ND
			100 μg/mL	ND	ND
			90 μg/mL	ND	ND
			80 μg/mL	ND	ND
			70 μg/mL	ND	ND
			60 μg/mL	ND	ND
			50 μg/mL	ND	ND
			40 μg/mL	89/384	0.91375
			30 μg/mL	98/384	0.85355
			20 μg/mL	87/384	0.92796
			10 μg/mL	100/384	0.84092
			5 μg/mL	74/384	1.02911
			Solvent control	92/384	0.89303
			Untreated control	90/384	0.90677
			Positive control (NQO)	121/384	0.72178

A2 = Assay 2

+ = in the presence of S9-mix

- = in the absence of S9-mix

CP = Cyclophosphamide (4 μg/mL)

NQO: 4-Nitroquinoline-N-oxide (0.1 μg/mL)

Solvent control = 1% (v/v) DMSO

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed during the treatment or in the expression period.

Table 12. Mutagenicity Results - Assay 2 (I.).

(3 hour treatment in the presence of S9-mix)

S 9 mix	Treatment period (hours)	Test item or control concentration	Number of empty wells / total number of wells	Number of large colonies / total number of wells	Number of small colonies / total number of wells	Dn^2/var(Dn) ♦	Mutation frequency
+	3	275 μg/mL	ND	ND	ND	ND	ND
		250 μg/mL	ND	ND	ND	ND	ND
		225 μg/mL	ND	ND	ND	ND	ND
		200 μg/mL	ND	ND	ND	ND	ND
		150 μg/mL	ND	ND	ND	ND	ND
		137.5 μg/mL	ND	ND	ND	ND	ND
		125 μg/mL	678/768	58/768	32/768	0.332298	69.3
		112.5 μg/mL	657/768	57/768	54/768	1.165325	79.0
		100 μg/mL	679/768	68/768	21/768	0.137574	65.3
		75 μg/mL	693/768	54/768	21/768	0.065262	63.4
		50 μg/mL	693/768	54/768	21/768	0.408148	48.7
		25 μg/mL	665/768	64/768	39/768	0.199861	66.5
		10 μg/mL	692/768	50/768	26/768	0.681770	46.1
		5 μg/mL	689/768	54/768	25/768	0.023125	56.2
		Untreated control	684/768	56/768	28/768	-	55.4
		Solvent control	685/768	55/768	28/768	-	58.8
		Positive control(CP: 4 μg/mL)	443/768	161/768	164/768	♦♦ 2.82E-11*	1054.9*

In linear trend analysis β²/var (β) = 2.65, not significant.

* = Statistically significant compared to the solvent control.

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn²/var(Dn) > 5.66 (at p<0.05).

♦♦ = Evaluated by T-test for independent samples. Significant at p<0.05 compared to the untreated control.

Dn = Difference of log mutant frequency of dose “n” and that of the vehicle control

var(Dn) = variance of Dn; β = slope of the curve; var(β) = variance of the slope

Solvent control = 1% (v/v) DMSO

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed in the expression period.

Table 13. Mutagenicity Results - Assay 2 (II.).

(24 hour treatment in the absence of S9-mix)

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells / total number of wells	Number of large colonies / total number of wells	Number of small colonies / total number of wells	Dn2/var(Dn) ♦	Mutation frequency
-	24	120 μg/mL	ND	ND	ND	ND	ND
		110 μg/mL	ND	ND	ND	ND	ND
		100 μg/mL	ND	ND	ND	ND	ND
		90 μg/mL	ND	ND	ND	ND	ND
		80 μg/mL	ND	ND	ND	ND	ND
		70 μg/mL	ND	ND	ND	ND	ND
		60 μg/mL	ND	ND	ND	ND	ND
		50 μg/mL	ND	ND	ND	ND	ND
		40 μg/mL	684/768	59/768	25/768	0.991151	63.4
		30 μg/mL	727/768	32/768	9/768	1.204367	32.1
		20 μg/mL	710/768	53/768	5/768	0.113249	42.3
		10 μg/mL	713/768	32/768	23/768	0.039570	44.2
		5 μg/mL	706/768	38/768	24/768	0.201687	40.9
		Untreated control	712/768	40/768	16/768	-	41.7
		Solvent control	706/768	42/768	20/768	-	47.1
		Positive control (NQO: 0.1 μg/mL)	329/768	338/768	101/768	♦♦ 9.69E-13*	587.3*

In linear trend analysis β²/var (β) = 0.04, not significant.

* = Statistically significant compared to the solvent control.

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn²/var(Dn) > 4.97 (at p<0.05).

♦♦ = Evaluated by T-test for independent samples. Significant at p<0.05 compared to the untreated control.

Dn = Difference of log mutant frequency of dose “n” and that of the vehicle control

var(Dn) = variance of Dn; β = slope of the curve; var(β) = variance of the slope

Solvent control = 1% (v/v) DMSO

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed during the treatment or in the expression period.

Table 14. Survival Results - Assay 3.

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells / total number of wells	Plating Efficiency (PE)	Harmonised Relative Survival (%) ^§ (%RS)
-	24	A3	120 μg/mL	ND	ND	ND
			110 μg/mL	ND	ND	ND
			100 μg/mL	ND	ND	ND
			90 μg/mL	ND	ND	ND
			80 μg/mL	ND	ND	ND
			70 μg/mL	ND	ND	ND
			60 μg/mL	ND	ND	ND
			50 μg/mL	134/384	0.65800	30
			45 μg/mL	105/384	0.81043	67
			40 μg/mL	148/384	0.59589	103
			30 μg/mL	153/384	0.57513	145
			20 μg/mL	171/384	0.50561	131
			10 μg/mL	161/384	0.54327	119
			5 μg/mL	169/384	0.51296	111
			Solvent control	169/384	0.51296	100
			Untreated control	189/384	0.44306	86
			Positive control (NQO)	295/384	0.16479	27

A3 = Assay 3

- = in the absence of S9-mix

NQO: 4-Nitroquinoline-N-oxide (0.1 μg/mL)

Solvent control = 1% (v/v) DMSO

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed during treatment.

Table 15. Viability Results - Assay 3.

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells / total number of wells	Plating Efficiency (PE)
-	24	A3	120 μg/mL	ND	ND
			110 μg/mL	ND	ND
			100 μg/mL	ND	ND
			90 μg/mL	ND	ND
			80 μg/mL	ND	ND
			70 μg/mL	ND	ND
			60 μg/mL	ND	ND
			50 μg/mL	118/384	0.73747
			45 μg/mL	107/384	0.79863
			40 μg/mL	104/384	0.81641
			30 μg/mL	122/384	0.71664
			20 μg/mL	107/384	0.79863
			10 μg/mL	104/384	0.81641
			5 μg/mL	100/384	0.84092
			Solvent control	128/384	0.68663
			Untreated control	110/384	0.78135
			Positive control (NQO)	115/384	0.75357

A3 = Assay 3

- = in the absence of S9-mix

NQO: 4-Nitroquinoline-N-oxide (0.1 μg/mL)

Solvent control = 1% (v/v) DMSO

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed during the treatment.

Table 16. Mutagenicity Results - Assay 3.

(24 hour treatment in the absence of S9-mix)

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells / total number of wells	Number of large colonies / total number of wells	Number of small colonies / total number of wells	Dn2/var(Dn) ♦	Mutation frequency
-	24	120 μg/mL	ND	ND	ND	ND	ND
		110 μg/mL	ND	ND	ND	ND	ND
		100 μg/mL	ND	ND	ND	ND	ND
		90 μg/mL	ND	ND	ND	ND	ND
		80 μg/mL	ND	ND	ND	ND	ND
		70 μg/mL	ND	ND	ND	ND	ND
		60 μg/mL	ND	ND	ND	ND	ND
		50 μg/mL	680/768	67/768	21/768	1.349840	82.5
		45 μg/mL	706/768	50/768	12/768	0.096294	52.7
		40 μg/mL	699/768	49/768	20/768	0.000927	57.7
		30 μg/mL	708/768	51/768	9/768	0.006123	56.8
		20 μg/mL	685/768	60/768	23/768	0.467819	72.5
		10 μg/mL	707/768	50/768	11/768	0.185382	50.7
		5 μg/mL	708/768	44/768	16/768	0.329832	48.4
		Untreated control	682/768	62/768	24/768	-	76.0
		Solvent control	709/768	38/768	21/768	-	58.2
		Positive control (NQO: 0.1 μg/mL)	314/768	375/768	79/768	♦♦ 6.39E-14*	593.4*

In linear trend analysis β²/var (β) = 0.86, not significant.

* = Statistically significant compared to the solvent control.

♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn²/var(Dn) > 5.48 (at p<0.05).

♦♦ = Evaluated by T-test for independent samples. Significant at p<0.05 compared to the untreated control.

Dn = Difference of log mutant frequency of dose “n” and that of the vehicle control

var(Dn) = variance of Dn; β = slope of the curve; var(β) = variance of the slope

Solvent control = 1% (v/v) DMSO

ND = No cells were plated for colony growing, due to excessive cytotoxicity observed during the treatment.

Conclusions:

No mutagenic effect of tea tree oil nor any formed metabolites was observed either in the presence or absence of a metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

A GLP-compliant in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells in accordance with OECD 476 to test the potential of tea tree oil (TTO) to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Treatment concentrations for the mutation assay were selected based on the results of a preliminary cytotoxicity test. Dimethyl sulfoxide was used as the co-solvent in this study.

In Assay 1, TTO was tested at concentrations of 275; 250; 225; 200; 150; 100; 75; 50; 25; 10 and 5 μg/mL during a 3-hour treatment with metabolic activation and at concentrations of 120; 110; 100; 90; 80; 70; 60; 40; 20; 10 and 5 μg/mL during a 3-hour treatment without metabolic activation. Following the treatment with metabolic activation, excessive cytotoxicity was observed at concentrations of 275, 250, 225, 200 and 150 μg/mL. Cells were therefore evaluated at concentrations of 100, 75, 50, 25, 10 and 5 μg/mL. Following the treatment without metabolic activation, excessive cytotoxicity was observed at concentrations of 120, 110, 100, 90 and 80 μg/mL. Cells were therefore evaluated at concentrations of 70, 60, 40, 20, 10 and 5 μg/mL. No significant increase in the mutation frequency was observed at the evaluated concentrations in either the presence or absence of metabolic activation. No significant dose response to the treatment was indicated by the linear trend analyses.

In Assay 2, TTO was tested at concentrations of 275; 250; 225; 200; 150; 137.5; 125; 112.5; 100; 75; 50; 25; 10 and 5 μg/mL during a 3-hour treatment with metabolic activation and at concentrations of 120; 110; 100; 90; 80; 70; 60; 50; 40; 30; 20; 10 and 5 μg/mL during a 24-hour treatment without metabolic activation. In the 3-hour treatment with metabolic activation, excessive cytotoxicity was observed at concentrations of 275, 250, 225, 200 and 150 μg/mL. Three additional treatment levels (137.5, 125 and 112.5 μg/mL) were included in order to fulfil the requirements regarding the acceptable level of cytotoxicity. Marked cytotoxicity was subsequently observed at 137.5 μg/mL. Cells were therefore evaluated at concentrations of of 125, 112.5, 100, 75, 50, 25, 10 and 5 μg/mL. In the 24 -hour treatment without metabolic activation, excessive cytotoxicity was observed at concentrations of 120, 110, 100, 90, 80, 70 and 60 μg/mL. An evaluation was therefore made at concentrations of 40, 30, 20, 10 and 5 μg/mL. No significant increase in mutation frequency was observed at the evaluated concentrations for either treatment time, whether in the presence or absence of metabolic activation. No significant dose response to treatment with TTO was indicated by linear trend analysis.

In Assay 3, TTO was tested at concentrations of 120, 110, 100, 90, 80, 70, 60, 50, 45, 40, 30, 20, 10 and 5 μg/mL during a 24-hour treatment without metabolic activation. Excessive cytotoxicity was observed at concentrations of 120, 110, 100, 90, 80, 70 and 60 μg/mL. An evaluation was therefore made at concentrations of 50, 45, 40, 30, 20, 10 and 5 μg/mL. No significant increase in mutation frequency was observed and no significant dose response to the treatment was indicated by linear trend analysis.

No precipitation of the test item was observed visually in any mutation experiment at the start or end of treatment.

In conclusion, no mutagenic effect of tea tree oil nor any formed metabolites was observed either in the presence or absence of a metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: A sample of Tea Tree Oil was tested for activity in the Salmonella typhimurium histidine mutation induced reversion assay for potential mutagenicity or carcinogenicity. (The Ames test)
- Short description of test conditions: The Ames test is an internationally validated short-term in vitro test to detect mutagenic activity in substances. The indicator organisms, test procedures, control procedures, media and reagents, quality assurance measures and evaluation criteria used were all as laid down in Maron, D.M. and Ames, B.N., 1983: "Revised methods for the Salmonella mutagenicity test." Mutation Research, vol. 113, pp. 173-215.
- Parameters analysed / observed: Observation of the ability or inability of S. typhimurium exposed to the test substance to grow on a histidine-free medium.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The sample was designated "Tea Tree Oil", in a container bearing no other relevant identifying marks.

Target gene:

The histidine operon.

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 102.

Additional strain / cell type characteristics:

other: histidine-requiring

Metabolic activation:

with and without

Metabolic activation system:

A commercial rat liver microsomal (S-9) preparation was used (see 'Any other information on materials and methods').

Test concentrations with justification for top dose:

10, 25, 50, 100 and 150 μl (with and without metabolic activation). Only results for doses < 50 µl were accepted in the final interpretation of the data, as Tea Tree Oil had significant actibacterial activity at higher doses.

Vehicle / solvent:

Serial two-fold dilutions from 10 mg per ml were prepared in dimethyl sulphoxide (DMSO).

Positive controls:

yes

Remarks:

For information on controls, see "Any other information on materials and methods".

Positive control substance:

other: Without S9 mix: Sodium azide; 2,4-DNPH. With S9 mix: 2-aminoacridine; 2-AAF; 2-aminofluorene, MMS.

Remarks:

For information on controls, see "Any other information on materials and methods".

Details on test system and experimental conditions:

- Preincubation: Test strains were cultivated in OXOID Nutrient Broth No. 2 prior to the assay.
- Media: Base agar consisted of Vogel and Bonner medium E containing 2% w/v glucose and 1.5% w/v OXOID Agar No. 3, dispensed as 25ml volumes in 90 mm petri dishes. Overlay agar contained 0.6% w/v agar, 0.5% w/v NaCl and 0.05mM each of L-histidine and D-biotin.
- Assay of dilution series in DMSO: The pre-incubation plate-incorporation assay, with and without metabolic activation, was employed, as described by Maron and Ames. Briefly, doses of 10, 25, 50, 100 and 150 μl of each dilution were mixed with 100 μl indicator culture and 500 μl of either 0.2M phosphate buffer, pH 7.4, or S-9 mix, and held at 37°C for 20 minutes. Then 2ml overlay agar was added, and the mixture poured over the surface of base agar plates. Once the overlay had set, plates were incubated at 37 ± 0.3°C for 48 hours. Each dose was examined in triplicate, and each assay was repeated at least once prior to processing the data.

Evaluation criteria:

The Ames Test is accepted to be only a semiquantitative indicator of mutagenicity and potential carcinogenicity. Accordingly, the criteria used to determine positive effects are inherently subjective, and must be based primarily on a historical data base. This data base is, however, sufficiently substantial to support the following criteria:

Given that:

1: there is no evidence of toxicity towards the indicator lawn, and the appropriate number of viable cells (1-2 x 10^8) are exposed to the sample;
2: the spontaneous background His+ reversion rate is consistently within the following normal limits: TA 98: 30-50, TA 100: 120-200, TA 102: 240-320 (values in the presence of S-9 may be up to 50% higher);
3: any increase in the number of revertants in the presence of test material is dose-dependant over at least part of the dose range examined;

then any sample giving a revertant count which is more than two standard deviations from the mean spontaneous count must be suspect, and any sample giving counts at least twice the spontaneous rate should be considered positive as a mutagen for these indicator strains in the Ames Test.

Statistics:

The data obtained in these assays consists of a series of replicate plate counts, from which mean count ±SD for each control and sample dose can be determined. Where the sample mean at any dose was not significantly different (see 'Evaluation Criteria' above) from the mean spontaneous background reversion rate, the sample as a whole was simply scored as "-". However, where any dose of a series from the sample gave a significantly elevated reversion (or forward mutation) count, the mean count ±SD for each dose was tabulated.

Species / strain:

other: S. typhimurium: TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Positive controls validity:

valid

Additional information on results:

Application of the Spot (disc) Test to a dilution series of the test substance in DMSO demonstrated that while histidine-like growth promoting activity was absent, the substance produced inhibition zones of increasing size as doses were increased above 50μg. Accordingly, only results for doses less than 50μg were accepted in the final interpretation of the data from the plate incorporation assay. Nevertheless, no elevation of revertant frequency was observed for doses up to 800μg in the latter assay.

Remarks on result:

other: all strains/cell types tested

His⁺Reversion Assay Controls:

Table 2. Spontaneous reversion rates.

Indicator Strain	His^+Revertants / plate (Mean ± SD)
	Without S-9	With S-9
TA 98	37 ± 3	48 ± 4
TA 100	153 ± 8	198 ± 11
TA 102	266 ± 10	331 ± 11

 

 

Table 3. Responses to Diagnostic Mutagens.

Mutagen		Strain Tested	His^+revertants / plate at dose (μl)
			10	25	50	100	150
Without S-9:	Sodium azide	TA 100	360 ± 15	795 ± 47	1502 ± 97	>2500	++++
	2,4-dinitrophenylhydrazine	TA 98	116 ± 6	235 ± 17	337 ± 12	650 ± 14	900 ± 31
		TA 102	436 ± 17	906 ± 27	1770 ± 31	++++	++++
With S-9:	2-aminoacridine	TA 100	417 ± 20	710 ± 11	1031 ± 47	1650 ± 50	++++
	2-acetamidofluorene	TA 98	293 ± 11	761 ± 17	1884 ± 72	>2200	++++
		TA 100	377 ± 13	696 ± 14	1158 ± 36	>2000	++++
		TA 102	476 ± 12	982 ± 21	1913 ± 61	>3000	++++
	2-aminofluorene	TA 98	>3000	++++	++++	toxic	toxic
		TA 100	1981 ± 36	>3000	++++	++++	++++
	Methylmethane sulphonate (MMS)	TA 102	>3000	++++	++++	toxic	toxic

[++++: uncountable semi-confluent growth of revertants]

Table 4. Detection Sensitivities of Indicators for Diagnostic Mutagens: (by graphic extrapolation of the data in Tables 2 and 3)

Mutagen Used	Indicator Strain	Dose (μg) required to give
		M.S.R.R* + 3 SD	2 x M.S.R.R
Sodium azide	TA 100	0.6	1.2
2,4-dinitrophenylhydrazine	TA 98	30	60
	TA 102	70	150
2-aminoacridine	TA 100	35	90
2-acetamidofluorene	TA 98	25	40
	TA 100	45	120
	TA 102	80	150
2-aminofluorene	TA 98	<1	<1
	TA 100	<2	<2
Methylmethane sulphonate	TA 102	<0.1	<0.1

*: M.S.R.R = Mean Spontaneous Reversion Rate

SD = Standard Deviation

Results obtained with Test Substance

Table 5: Summary of Data Obtained with Tea Tree Oil.

Indicator	Mutagenic activity
	Without metabolic activation	With metabolic activation (S-9)
TA 98	-*	-
TA 100	-	-
TA 102	-	-

*: No apparent dose dependence in the frequency of revertants, and no counts more than 2 standard deviations greater than the mean spontaneous reversion rate for the indicator strain under the appropriate conditions (see Table 2).

The mean number of revertant colonies per plate is not noted for the different test concentrations.

Interpretation of the results obtained in this study is complicated by the fact that the test substance has significant antibacterial activity at doses above 50μg. Further, the volatility and low aqueous solubility of this essential oil make it difficult to assess the carcinogenic hazard posed by this substance. Nevertheless, the results do suggest that Tea Tree Oil is not active as an inducer of reversion in the Ames panel of Salmonella typhimurium His^- mutants at doses below 50μg and it is reasonable to assert from the results of this study that Tea Tree Oil is unlikely to constitute a carcinogenic hazard.

Conclusions:

Tea Tree Oil does not have mutagenic potential.

Executive summary:

A sample of Tea Tree Oil was tested for activity in the Salmonella typhimurium histidine mutation induced reversion assay for potential mutagenicity or carcinogenicity (the Ames Test). The Ames test panel included strains of S. typhimurium known to be particularly susceptible to DNA frameshifting agents, to agents which cause DNA base substitutions, and to agents which alkylate or cross-link DNA. Although Tea Tree Oil has marked antibacterial properties, at doses of 50 μg or less it lacked toxicity and was devoid of reversion-inducing activity towards the entire panel of indicator organisms used in either the presence or absence of S-9 mix. This substance can therefore be inferred to lack any mutagenic or carcinogenic activity demonstratable by the Salmonella typhimurium histidine mutation induced reversion assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The mutagenicity of tea tree oil has been evaluated using a bacterial reverse mutation assay in S. typhimurium (Bolt, 1989), an in vitro gene mutation study in mammalian cells (Hargitai, 2010) and an in vitro cytogenicity / chromosome aberration study in mammalian cells (Beres, 2009).  A negative result was obtained in each study.   Based on CLP/GHS criteria, it is concluded that tea tree oil should not be classified on the basis of mutagenicity.