Melaleuca alternifolia, ext.

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Test guideline 3

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.

Test material

Test material information

Constituent 1

Reference substance name:

Melaleuca alternifolia, ext.

EC Number:

285-377-1

EC Name:

Melaleuca alternifolia, ext.

Cas Number:

85085-48-9

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).

IUPAC Name:

Essential oil of melaleuca alternifolia

Test material form:

liquid

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.

Method

Species / strain

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.

Controls

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).

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.