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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Monitoring Programme (inspected between 2012-06-18 and 2012-06-20)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Name of test material (as cited in study report): eucalyptus oil globulus
- Molecular formula (if other than submission substance): see confidential
- Molecular weight (if other than submission substance): see confidential
- Smiles notation (if other than submission substance): see confidential
- Structural formula attached as image file (if other than submission substance): see confidential
- Substance type: Natural Complex Substance
- Physical state: see confidential
- Analytical purity: see confidential
- Impurities (identity and concentrations): see confidential
- Composition of test material, percentage of components: see confidential
- Purity test date: see confidential
- Lot/batch No.: see confidential
- Expiration date of the lot/batch: see confidential
- Storage condition of test material: room temperature
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

Method

Target gene:
His+ for S. typhimurium; trp+ for E. coli
Species / strain
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.
Controls
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.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

See attached Document for Tables of Results

Applicant's summary and conclusion

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.