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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:
10-03-2017 to 27-03-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: ISO/IEC 17025:2005
Version / remarks:
2005
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Clear whitish-yellow liquid (per BioReliance)
Details on test material:
Identification: Ylang Oil I
RIFM Identification Number: 6614-F2.12.1
CAS No.: 8006-81-3; Alternate CAS No.: 83863-30-3
Purity: Not provided
Molecular Weight: Not Provided
Description: Clear whitish-yellow liquid (per BioReliance)
Essential Oil obtained by steam distillation first grade from flower (per Protocol)
Storage Conditions: Room temperature, protected from light
Receipt Date: 01 February 2017

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Obtained from sponsor
- Expiration date of the lot/batch: 26-01-2019
- Purity test date: 27-01-2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

OTHER SPECIFICS: Clear whitish-yellow liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First mutation experiment: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate
Second mutation experiment: 15.0, 50.0, 150, 500, 1500, 3333 and 5000 μg/plate (TA98) and 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate (other conditions)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incorporation assay
- Cell density at seeding (if applicable): 0.3x10^9 cells per milliliter

DURATION
- Exposure duration: 48 to 72 hours at 37±2°C.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies.

OTHER: The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test substance to be evaluated positive, it must cause a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
- Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative if it is neither positive nor equivocal.

Results and discussion

Test results
Key result
Species / strain:
other: TA98, TA100, TA1535 and TA1537 and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitate was observed at 5000 μg per plate with all conditions
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
-First mutation experiment: No toxicity was observed. Precipitate was observed at 5000 μg per plate with all conditions. A non-dose responsive increase (1.6- fold, maximum increase) was observed with tester strain WP2 uvrA in the absence of S9 activation (within the 95% HCL). A 1.5- fold, maximum increase was also observed with tester strain TA98 in the presence of S9 activation (Outside the 95% HCL).
-Second mutation experiment: Precipitate was observed at 5000 μg per plate with all conditions. No background lawn toxicity was observed; however a reduction in revertant count was observed at 5000 μg per plate with tester strain TA1535 in the presence of S9 activation. A non-dose responsive increase of 1.5-fold, maximum increase was observed with tester strain TA98 in the presence of S9 activation. While the average is one colony outside the 95% HCL, the increase was not dose responsive and there is a lot of variability with the individual plate counts. Therefore, this response is non-mutagenic.

HISTORICAL CONTROL DATA
- Positive historical control data: All tester strain cultures were within ranges of historical control values (2015).
- Negative (solvent/vehicle) historical control data: All tester strain cultures were within ranges of historical control values (2015).

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, it is concluded that Ylang Ylang I is not mutagenic and does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The mutagenic potential of Ylang Oil I was evaluated according to guideline OECDTG 471. Ylang Oil I was tested in Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100 and Escherichia coli tester strain WP2uvr at concentrations of 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate in the absence and presence of S9-mix. Based on the first mutation experiment, the following dose-range was selected for the second mutation experiment with the Salmonella typhimurium tester strains TA98 in the absence and presence of S9-mix: 15.0, 50.0, 150, 500, 1500, 3333 and 5000 μg/plate, and 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate with all other conditions. Precipitation of Ylang Oil I on the plates was observed at 5000 μg/plate with all conditions. Cytotoxicity, as evidenced by a decrease in the number of revertants, was not observed. Ylang Oil I did not induce a significant dose-related increase in the number of revertant (His +) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2 uvrA, both in the absence and presence of S9-metabolic activation. In this study, acceptable responses were obtained for the negative and strainspecific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. It is concluded that Ylang Ylang I is not mutagenic and does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).