Eucalyptus globulus, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).
- ML/TK test: non mutagenic (OECD 476, GLP, K, rel. 1).
- HL CAT: not clastogenic (OECD 473, GLP, K, rel.1).

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:

24 October - 13 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Monitoring Programme (inspected between 2012-06-18 and 2012-06-20)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Eucalyptus oil (Eucalyptus globulus, ext)
- Physical state: Yellow to pale yellow liquid
- Analytical purity: 100%
- Lot/batch No.: 0712F08
- Date of receipt: 26 September 2012
- Expiration date of the lot/batch: 3 August 2014
- Storage condition of test material: At ambient temperature protected from air and light

Target gene:

His+ for S. typhimurium; trp+ for E. coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Phenobarbital sodium/5,6-benzoflavone.

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains
Experiment 2 (pre-incubation method): 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Dimethyl sulphoxide)
- Justification for choice of solvent/vehicle: The solubility of Eucalyptus Oil was assessed at 50 mg/mL in DMSO and in acetone. It was found to be soluble in both solvents. DMSO is, however, relatively less toxic towards the tester strains and was, therefore, used as the vehicle for this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.2.1/1

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Strains of S. typhimurium and E. coli were obtained from Moltox Inc.

METHOD OF APPLICATION:
Experiment 1: In agar (direct plate incorporation)
Experiment 2: preincubation method

DURATION
- Preincubation period: Exp. 2, 30 minutes at 37 °C
- Incubation period: Approximately 72 h at 37 °C for both direct plate incorporation and preincubation methods

NUMBER OF REPLICATIONS:
-3 plates/dose for treatment, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.

OTHER: After 72 h of incubation at 37 °C, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

Evaluation criteria:

- If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
- If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
- If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
- Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.

Statistics:

- Statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none seen
- Other confounding effects: none

CYTOTOXICITY:
- Experiment 1: No evidence of toxicity was observed.
- Experiment 2: Toxicity, observed as a reduction in revertant colony numbers, was obtained in all strains following exposure to Eucalyptus Oil at 5000 µg/plate in the absence of S9 mix. No evidence of toxicity was obtained in the presence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical data from the period 1 May 2007 to 30 April 2012.

OTHERS:
- Viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.

See attached Document for Tables of Results

Conclusions:

Under the test conditions, Eucalyptus oil is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains ofSalmonella typhimurium(TA 1535, TA 1537, TA 98 and TA 100) andEscherichia coli (WP2uvrA)were exposed to Eucalyptus oil at the following concentrations:

Experiment 1 (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains

Experiment 2 (pre-incubation method): 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains

 

Metabolic activation system used in this test was10 % (v/v) S9 mix;S9 fraction prepared from liver homogenates of rats induced with Phenobarbital sodium/5,6-benzoflavone.Vehicle and positive control groups were also included in mutagenicity tests.

 

No signs of toxicity towards the tester strains were observed in the first experiment following exposure to test item. Toxicity, observed as a reduction in revertant colony numbers, was obtained in all strains in the second experiment following exposure to test item at 5000 µg/plate in the absence of S9 mix. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Eucalyptus Oil at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

 

Under the test conditions,Eucalyptus oilis not considered as mutagenic in these bacterial systems.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October - 30 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD 473 Guideline without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: human lymphocytes

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

5% v/v (final concentration; S9 fraction was obtained from the liver of rats treated with Phenobarbital sodium and 5,6 Benzoflavone by intraperitoneal route.

Test concentrations with justification for top dose:

Preliminary toxicity test:
- With and without S9 mix (3 h treatment and 18 h recovery): 139.97, 233.28, 388.8, 648, 1080, 1800, 3000 and 5000 µg/mL
- Without S9 mix (21 h continuous treatment): 139.97, 233.28, 388.8, 648, 1080, 1800, 3000 and 5000 µg/mL
- With and without S9 mix (3 h treatment and 18 h recovery): 4.03, 6.72, 11.2, 18.66, 31.1, 51.84, 86.4, 144, 240 and 400 µg/mL
- Without S9 mix (21 h continuous treatment): 4.03, 6.72, 11.2, 18.66, 31.1, 51.84, 86.4, 144, 240 and 400 µg/mL

Main test:
- Without S9 mix (3 h treatment and 18 h recovery): 10, 20, 40, 60, 80 and 1000 µg/mL
- With S9 mix (3 h treatment and 18 h recovery): 100, 150, 200, 250, 275, 300, 325 and 350 µg/mL
- Without S9 mix (21 h continuous treatment): 50, 60, 70, 80, 90, 100, 110 and 120 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Formulation preparation: Eucalyptus oil was found to be miscible in acetone at 500 mg/mL (1M). On dosing a 500 mg/mL solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of 5000 µg/mL, the test compound was visible within the medium, however evenly distributed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.2 µg/mL (3 hour treatment) and 0.1 µg/mL (21 hour continuous treatment); without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

5 µg/mL (3 hour treatment); with metabolic activation

Details on test system and experimental conditions:

PREPARATION OF CULTURES:
- Human blood was collected aseptically from two healthy, non-smoking, adult donors, pooled (in equal volumes from each donor) and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM L-glutamine. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each contained blood (0.4 mL), culture medium (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 37 °C, and the cells resuspended (at least once daily) by gentle inversion.

DURATION
- Exposure duration: Preliminary toxicity test: 3 h (±S9); 21 h continuous treatment (-S9); Main test: 3 h (±S9); 21 h continuous treatment
- Fixation time (start of exposure up to harvest of cells): 21 h, with and without S9 mix in preliminary toxicity and main tests

SPINDLE INHIBITOR (cytogenetic assays): Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 0.1 µg/mL.

STAIN (for cytogenetic assays): Giemsa staining (10% in buffered water (pH 6.8))

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment with test substance for each test condition.
- Main test: Duplicate cultures were used for treatment with the vehicle, test substance and positive controls.

NUMBER OF CELLS EVALUATED:
- Cytotoxicity of the test item was evaluated using the mitotic index (% cells in mitosis), which indicates whether an item induces mitotic inhibition. The mitotic index was determined in a sample of 1000 cells per culture of each test group.
- One hundred metaphase figures were examined from each culture, however, this number was reduced in cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed. The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) was recorded as a percentage of the 100 metaphases analysed per slide.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

Evaluation criteria:

An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
- Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
- The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
- The increases are reproducible between replicate cultures.
- The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
- Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Statistics:

- Number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).
- A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.
- D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002).

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No; No fluctuations in osmolality of more than 50 mOsm/kg or changes in pH of more than 1.0 unit were observed at 5000 μg/mL when compared with the vehicle control.

PRELIMINARY TOXICITY TEST:
- In the absence of S9 mix following 3 h treatment, Eucalyptus oil caused a reduction in the mitotic index to 25% of the vehicle control value at 139.97 µg/mL.
- In the presence of S9 mix following 3 h treatment, Eucalyptus oil caused a reduction in the mitotic index to 31% of the vehicle control value at 388.8 µg/mL.
- In the absence of S9 mix following 21 h treatment, Eucalyptus oil caused overt toxicity in all concentrations tested. As a result of overt toxicity in all treatment regimens an additional preliminary test was performed using lower concentrations to determine a toxicity profile for the main assay.
- In the absence of S9 mix following 3 h treatment, Eucalyptus oil caused a reduction in the mitotic index to 52% of the vehicle control value at 86.4 µg/mL.
- In the presence of S9 mix following 3 h treatment, Eucalyptus oil caused a reduction in the mitotic index to 72% of the vehicle control value at 240 µg/mL.
- In the absence of S9 mix following 21 h treatment, Eucalyptus oil caused a reduction in the mitotic index to 32% of the vehicle control value at 144 µg/mL.

MAIN TEST:
- 3 h treatment in the absence of S9 mix: Eucalyptus oil caused a reduction in the mitotic index to 51% of the vehicle control value at 100 µg/mL. The concentrations selected for metaphase analysis were 10, 80 and 100 µg/mL.
- 3 h treatment in the presence of S9 mix: Eucalyptus oil caused a reduction in the mitotic index to 51% of the vehicle control value at 350 µg/mL. The concentrations selected for metaphase analysis were 200, 325 and 350 µg/mL.
- 21 h treatment in the absence of S9 mix: Eucalyptus oil caused a reduction in the mitotic index to 47% of the vehicle control value at 90 µg/mL. The concentrations selected for metaphase analysis were 50, 60 and 90 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical data of the laboratory (October 2010 – September 2012).

Remarks on result:

other: other: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/1: Summary of results

Exposure period (h)	S9 mix	Nominal concentration of Eucalyptus Oil (µg/mL)	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative Mitotic
			Individual values (%)		Mean (%)	Individual values (%)		Mean (%)	Index  (%)
3	-	0 (Acetone)	3.0	4.0	3.5	5.0	10.0	7.5	100
		10	3.0	1.0	2.0	3.0	3.0	3.0	92
		80	2.0	2.0	2.0	5.0	6.0	5.5	70
		100	1.0	8.0	4.5	6.0	11.0	8.5	51
		0.2 (Mitomycin C)	18.9	15.6	17.1***	28.3	20.3	23.9***	-
3	+	0 (Acetone)	1.0	1.0	1.0	3.0	3.0	3.0	100
		200	1.0	6.0	3.5	3.0	8.0	5.5	106
		325	3.0	3.0	3.0	3.0	4.0	3.5	74
		350	1.0	5.0	3.0	3.0	9.0	6.0	51
		5 (Cyclophosphamide)	6.0	7.0	6.5**	10.0	10.0	10.0**	-
21	-	0 (Acetone)	0.0	3.0	1.5	2.0	5.0	3.5	100
		50	5.0	4.0	4.5	8.0	7.0	7.5	94
		60	3.0	2.0	2.5	8.0	5.0	6.5	72
		90	3.0	1.0	2.0	8.0	3.0	5.5	47
		0.1 (Mitomycin C)	37.0	30.3	33.3***	40.7	39.4	40.0***	-

One-tailed Fisher's exact test-***:p<0.001; **:p<0.01; Otherwise p>0.01

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the test conditions, Eucalyptus oil is not considered as clastogenic in cultured human lymphocytes with and without metabolic activation.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to Eucalyptus oil at the following concentrations:

 

Preliminary toxicity test:

- With and without S9 mix (3 h treatment and 18 h recovery): 139.97, 233.28, 388.8, 648, 1080, 1800, 3000 and 5000 µg/mL

- Without S9 mix (21 h continuous treatment): 139.97, 233.28, 388.8, 648, 1080, 1800, 3000 and 5000 µg/mL

- With and without S9 mix (3 h treatment and 18 h recovery): 4.03, 6.72, 11.2, 18.66, 31.1, 51.84, 86.4, 144, 240 and 400 µg/mL

- Without S9 mix (21 h continuous treatment): 4.03, 6.72, 11.2, 18.66, 31.1, 51.84, 86.4, 144, 240 and 400 µg/mL

 

Main test:

- Without S9 mix (3 h treatment and 18 h recovery): 10, 20, 40, 60, 80 and 1000 µg/mL

- With S9 mix (3 h treatment and 18 h recovery): 100, 150, 200, 250, 275, 300, 325 and 350 µg/mL

- Without S9 mix (21 h continuous treatment): 50, 60, 70, 80, 90, 100, 110 and 120 µg/mL

 

Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 0.1 µg/mL. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Metabolic activation system used in this test was 5% v/v (final concentration; S9 fraction was obtained from the liver of rats treated with Phenobarbital sodium and 5,6 Benzoflavone by intraperitoneal route.

 

Cytotoxicity was observed in various concentrations and doses were selected based on the mitotic index data. No statistically significant increases in the chromosomal aberrations, polyploid or endoreduplicated metaphase cells were observed under any treatment condition at any analysed concentration, with or without metabolic activation, when compared to the vehicle control. The positive control substances (mitomycin-C; without S9 mix and cyclophosphamide; with S9 mix) caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

 

Under the test conditions, Eucalyptus oil is not considered as clastogenic in cultured human lymphocytes with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January - 25 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD Guideline 476 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source: American Type Culture Collection (ATCC), Virginia.
- Type and identity of media: RPMI 1640 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 h incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 °C, in heat-inactivated donor horse serum (HiDHS) containing 10 % DMSO.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (5% v/v); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6 benzoflavone.

Test concentrations with justification for top dose:

Preliminary toxicity test:
- 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL, with S9 mix (3 h exposure) and without S9 mix (3 and 24 h exposure)

Mutation tests:
- Without S9 mix (3 h exposure): 10, 100, 150, 200, 225, 250, 275 and 300 μg/mL
- Without S9 mix (3 h exposure, additional test): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- With S9 mix (3 h exposure): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- Without S9 mix (24 h exposure): 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Formulation preparation: Eucalyptus oil was dissolved and diluted in acetone (analytical grade), shortly before dosing. The final concentration of acetone added to the cultures was 1 % v/v. Eucalyptus oil was found to be miscible at 500 mg/mL in acetone.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

10 μg/mL (3 h exposure); 5 μg/mL (24 h exposure) - without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

1 μg/mL (3 h exposure); with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: RPMI 1640 medium
- R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
- R10p: R0, supplemented with 0.1 % v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10 % v/v.
- R30p: R0, supplemented with 0.02 % v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30 % v/v.
- R10p medium was used for cell culture unless otherwise specified.
- R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.

DURATION
- Exposure duration: Preliminary toxicity test: 3 and 24 h exposure (without S9 mix) and 3 h exposure (with S9 mix); Mutation tests: 3 and 24 h exposure (without S9 mix); 3 h exposure (with S9 mix)
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 7 days for viability plates and approximately 10 to 14 days for mutant plates
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

SELECTION AGENT (mutation assays): Selective medium consisted of R10p containing 4 μg/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and 2 cultures for vehicle control
- Main test: 4 cultures for vehicle control, 2 cultures/dose for test item and positive controls

NUMBER OF CELLS EVALUATED: 1.6 and 2000 cells per well plated for assessing cloning efficiency (CE) and mutant frequency (MF), respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG), Cloning efficiency (CE), Relative cloning efficiency (RCE) and Relative total growth (RTG)
RSG = (Individual SG x 100) / Mean vehicle control SG
CE = - InP(0) / No. of cells per well
RCE = (Individual CE x 100) / Mean vehicle control CE
RTG = (RSG x Day2 RCE) / 100

OTHER:
Mutant frequency per 10^6 survivors (MF) was calculated as:: CE selective medium / CE non-selective medium

Evaluation criteria:

The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10 %:
Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10^-6 (Moore et al., 2006).
The assay was considered valid in accordance with the assay acceptance criteria.
The test agent was regarded as negative if:
- The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.
If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
- If the linear trend test was negative, the result was regarded as negative.
- If the linear trend test was positive, this indicated a positive, biologically relevant response.
- Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
- Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001. Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in the pH of the medium of more than 1.0 unit compared with the vehicle control was observed at 5000 μg/mL.
- Effects of osmolality: The osmolality of the test substance in medium was tested at a concentration of 5000 μg/mL; no change in osmolality of the medium of more than 50 mOsm/kg was observed when compared with the vehicle control.
- Precipitation: A solution of 500 mg/mL, dosed at 1 % in medium, showed no precipitate in the culture medium.

PRELIMINARY TOXICITY TEST:
- In preliminary toxicity test, precipitate (observed by eye at the end of treatment) was observed at 312.5 μg/mL and above and 625 μg/mL and above in the absence and presence of S9 mix respectively, following a 3 h exposure. Exposure to Eucalyptus oil at concentrations from 9.77 to 5000 μg/mL in the absence and presence of S9 mix (3 h exposure) resulted in relative suspension growth (RSG) values between 99 and 0 % and between 91 and 0 % respectively. Following a 24 h exposure in the absence of S9 mix (from 9.77 to 5000 μg/mL), no precipitation (assessed by eye at the end of treatment) was observed at any concentration and RSG was reduced from 98 to 0 %.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data (14 March 2011 - 18 February 2013)

Remarks on result:

other: strain/cell type: TK^+/-

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/1: Results summary

Test Article	Dose Level µg/mL	3 h Treatment ‑S9‑mix		3 h Treatment +S9‑mix		24 h Treatment ‑S9‑mix
		Mean Relative Total Growth (%)	Mean Mutant Freq. (x10^‑6)	Mean Relative Total Growth (%)	Mean Mutant Freq. (x10^‑6)	Mean Relative Total Growth (%)	Mean Mutant Freq. (x10^‑6)
Acetone	0	100	86	100	75	100	61
Eucalyptus Oil	10	110	67	96	73	106	83
	50					77	80
	100					51	66
	115	95	74
	145	89	66	84	81
	150					13	68
	160	51	89
	175	18	84	76	93
	225			53	84
	250			22	117
Methyl methanesulphonate	10	128	699
	5					78	746
Benzo[a]pyrene	1			96	380

 

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, Eucalyptus oil is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk^+/-(3.7.2C) mouse lymphoma cells were exposed to Eucalyptus oil at the following concentrations:

 

Preliminary toxicity test:
- 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL, with S9 mix (3 h exposure) and without S9 mix (3 and 24 h exposure)

Mutation tests:
- Without S9 mix (3 h exposure): 10, 100, 150, 200, 225, 250, 275 and 300 μg/mL
- Without S9 mix (3 h exposure, additional test): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- With S9 mix (3 h exposure): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- Without S9 mix (24 h exposure): 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 μg/mL

 

Vehicle (acetone) and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 5 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6 benzoflavone.

 

In preliminary toxicity test, precipitate (observed by eye at the end of treatment) was observed at 312.5 μg/mL and above and 625 μg/mL and above in the absence and presence of S9 mix respectively, following a 3 h exposure. Exposure to Eucalyptus oil at concentrations from 9.77 to 5000 μg/mL in the absence and presence of S9 mix (3 h exposure) resulted in relative suspension growth (RSG) values between 99 and 0 % and between 91 and 0 % respectively. Following a 24 h exposure in the absence of S9 mix (from 9.77 to 5000 μg/mL), no precipitation (assessed by eye at the end of treatment) was observed at any concentration and RSG was reduced from 98 to 0 %. Following 3 h treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF) (212x 10^-6 and 201 x 10^-6, respectively), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 h treatment in the absence and presence of S9 mix were 175 and 250 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 18 and 22 % respectively. In the 24 h treatment, the maximum concentration assessed for mutant frequency was 150 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF (187 x 10^-6), within acceptable levels of toxicity. The RTG was reduced to 13 %. Mutant frequencies in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals [Methyl methanesulphonate (without S9 mix) and Benzo[a]pyrene (with S9 mix)] indicating the validity of the study.

 

Under the test conditions, Eucalyptus oil is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   May, 2013	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537 TA 98 TA 100 WP2 uvrA	-S9 +S9	Up to 5000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic
2   Sivaswamy, 1991	Ames Test D, rel. 3	Gene mutation	TA 1535, TA 1537 TA 98 TA 1538	-S9	Up to 10 pL/plate	-S9 : mutagenic
3   Woods, 2013	ML/TK test (OECD 476) K, rel. 1	Gene mutation	mouse lymphoma L5178Y cells	-S9 +S9	Up to cytotoxicity limit	-S9 : non mutagenic +S9 : non mutagenic
4   Woods, 2013	HL CAT (OECD 473) K, rel. 1	Chromosomal aberration	Human Lymphocytes	-S9 +S9	Up to cytotoxicity limit	-S9 : non clastogenic +S9 : non clastogenic

Gene mutation Assays (Tests n° 1-3):

Positive results were reported in the publication of Sivaswamy (1991)(Test n°2). However, the composition of the tested oil was not reported and the study did not meet important criteria of today’s standard method. Indeed, the test was conducted in the absence of metabolic activation only, without replicate and the selection of dose levels was not justified. Therefore the study was disregarded and a new test was performed (May, 2013)(Test n°1). This bacterial reverse mutation assay (Ames test), performed according to OECD 471 test guidelines and in compliance with GLP, was selected as the key study. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in both tests, with any dose of the test material, either in the presence or absence of metabolic activation. The test indicates that Eucalyptus oil does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. Eucalyptus oil is therefore considered as non-mutagenic according to the Ames test.

Inability to produce gene mutation was confirmed in mammals using an in vitro forward mutation assay in mouse lymphoma TK L5178Y cells (ML/TK test) (Test n°3). None of the dose levels up to the cytotoxicity limit with Eucalyptus oil, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. Eucalyptus oil does not induce forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Eucalyptus oil is therefore considered as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of both Ames tests and extends the non-mutagenic effect of Eucalyptus oil to mammalian cells.

Chromosomal aberration (Test n°4):

The clastogenic potential of the test material was determined using an in vitro chromosome aberration test in human lymphocytes, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes. None of the dose levels up to the cytotoxicity limit with Eucalyptus oil, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. Eucalyptus oil does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. Eucalyptus oil is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 including ATP3.

Self-classification:

Based on the available information, no additional classification is proposed.