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Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD TG 471): negative

Micronucleus human peripheral blood lymphocytes (OECD TG 487): negative

Gene mutation in mammalian cells (OECD TG 490): negative

Link to relevant study records

Referenceopen allclose all

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
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
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
Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
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
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.
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).
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).

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-03-2017 to 11-04-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
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
Species / strain / cell type:
lymphocytes: Human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healty non-smoking donor
- Sex, age and number of blood donors if applicable: one Male, 24 years
- Methods for maintenance in cell culture if applicable: lymphocytes were cultured in complete medium and incubated under standard conditions for 44-48 hours.

MEDIA USED
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B (CytoB)
Metabolic activation:
with and without
Metabolic activation system:
Aroclos 1254-induced rat liver S9
Test concentrations with justification for top dose:
Non-activated, treatment 4h, recovery 20h: 5, 10, 50, 75, 100, 125, 150, 250, 500 μg/mL
Non-activated, treatment 24h, recovery 0h: 1, 10, 100, 125, 150, 200, 300, 400, 500 μg/mL
S9-activated, treatment 4h, recovery 20h: 5, 10, 50, 75, 100, 125, 150, 250, 500 μg/mL
The highest dose evaluated for the micronuclei was selected based on a preliminary (cyto)toxicity test (CBPI relative to the vehicle control).
Vehicle / solvent:
- Vehicle(s) used: ethanol (CAS 64-17-5, Sigma-Aldrich)
- Justification for choice of solvent/vehicle: based on the solubility of the test substance and compatibility with the target cells
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Vinblastine and sterile water
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 44-48 hours
- Exposure duration: 4 hours in the absence and presence of S9 and 24 hours in the absence of S9, by incorporation of the test substance vehicle mixture into the treatment medium.
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells. The cyto B exposure time for the 4 hour treatment in the non-activated and the S9-activated studies was 20 hours (± 30 minutes).

STAIN: acridine orange

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange and identified by the BioReliance study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.

NUMBER OF CELLS EVALUATED: Cell cycle kinetics scoring: For the preliminary toxicity test, at least 500 cells, if possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture), if possible, were evaluated to determine the CBPI at each dose level and the control.
Micronucleus Scoring: Whenever possible, a minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- the micronucleus should have the same staining characteristics as the main nucleus
- the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
- the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity: ≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control.
- CBPI =1 x Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells/Total number of cells scored. % Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)} (t = test substance treatment culture, c = vehicle control culture)
Rationale for test conditions:
According to OECD TG 487
Evaluation criteria:
The test substance was considered to have induced a positive response if
- at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
- the increase was concentration-related (p ≤ 0.05), and
- results were outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Key result
Species / strain:
lymphocytes: Human peripheral blood lymphocytes
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:
TEST-SPECIFIC CONFOUNDING FACTORS
Visible precipitate and hemolysis were observed in treatment medium at the following doses:
- Effects of pH: The pH of the highest dose of test substance in treatment medium was 7.5.
- Effects of osmolality: The osmolality of the test substance doses in treatment medium in the preliminary toxicity test were considered acceptable.
- Precipitation: from 150 µg/mL for all treatment conditions

PRELIMINARY TEST: HPBL were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures (doses from 0.5 to 5000 μg/mL). Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, lowest precipitating dose, and the highest soluble dose was measured. Dose levels for the micronucleus assay were based upon post-treatment toxicity (CBPI relative to the vehicle control).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No significant or dose-dependent increases in micronuclei induction were observed in the treatment groups with and without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

HISTORICAL CONTROL DATA
The results for positive and negative controls for 4 hours in absence and presence of metabolic activation, and 24 hours in absence of metaboloc activation were in line with the available historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity (≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) was observed at doses ≥ 150 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses 150, 1500, and 5000 μg/mL in the S9-activated 4-hour exposure group:

Treatment Condition Treatment Time Highest Evaluated Dose (μg/mL) Cytotoxicity(%)
Non-activated 4 hr 125 56
S9-activated 4 hr 250 51
Non-activated 24 hr 125 55
Conclusions:
Under the conditions of the study, Ylang oil I was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes. Based on this result, the substance does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).
Executive summary:

Ylang oil I was tested for its potential to induce micronucleus formation in the in vitro micronucleus test with isolated human lymphocytes from fresh whole human blood, according to OECD TG 487. A preliminary toxicity study was performed with dose ranges from 0.5 to 5000 μg/mL. Cytotoxicity (≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) was observed. Based on these results, the doses chosen for the micronucleus assay ranged from 5 to 500 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 1 to 500 μg/mL for the non-activated 24-hour exposure group. The S9 fraction was obtained from Aroclor 1254-induced rat liver. Ethanol was used as a vehicle in the test system. Visible precipitation and hemolysis were observed under all treatment conditions.

Cytokinesis of the cells was blocked using cytochalasin B. Calculation of the Cytokinesis Block Proliferation Index (CBPI; at least 500 cells from appropriate cultures were scored) was used to demonstrate cell division and in order to select doses to process and score microscopically for micronuclei. For the selected doses, the micronucleus frequency was determined from 1000 from each culture (2000 binucleated cells per concentration). The results for positive and negative controls indicate that all criteria for a valid assay were met. No significant or dose-dependent increases in micronuclei induction were observed in the treatment groups with and without S9. Under the conditions of the assay described in this report, Ylang oil I was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes. Based on this result, the substance does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31-07-2017 to 11-11-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
The thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: L5178Y/TK+/--3.7.2C mouse lymphoma cells, Recommended test system in international guidelines (e.g.OECD).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Horse serum inactivated by incubation at 56°C for at least 30 minutes/ Basic medium RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin /Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium) / Exposure medium: For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium) / Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine (TFT) (Sigma) / Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Without S9-mix: 0.63, 1.25, 2.5, 5, 10, 20, 30, 40, 50, 60, 70 and 80 μg/ml exposure medium.
With S9-mix: 2.5, 5, 10, 20, 30, 45, 60, 70, 80, 90, 100 and 110 μg/ml exposure medium.

In a dose-range finding test, both in absence and presence of S9- mix, no cell survival was observed at test item concentrations of 125 μg/ml and above.
The test item precipitated in the culture medium at dose levels of 250 μg/ml and above.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: according to guideline
Untreated negative controls:
yes
Remarks:
DSMO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium) (with and without metabolic activation). 24 hours: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium) (without metabolic activation).

Dose-range finding test:
- After exposure, cells were separated from treatment solutions and resuspended in growth medium. The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity. After 24 hour of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted. The surviving cells of the 24 hour treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 10^5 cells/ml were counted no subculture was performed. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests. The cells in the final suspension were counted.

Mutagenicity test:
- For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

STAIN: The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

NUMBER OF REPLICATIONS: only the solvent control was tested in duplicate.

NUMBER OF CELLS EVALUATED:
-For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
-For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity is identified as a reduction in relative total growth (RTG). The RTG was calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture: RTG = RSG x RCE/100

- OTHER: Analysis
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical
control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Rationale for test conditions:
According to OECD guideline.
Evaluation criteria:
- In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
- The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Key result
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test item precipitated directly in the exposure medium at concentrations of 78 μg/ml and above. After 3 hours, the test item precipitated in the exposure medium at concentrations of 625 μg/ml and above. In the dose-range finding test, the test item precipitated in the culture medium at dose levels of 250 μg/ml and above.
- Other confounding effects: In the dose range finding test, no cell survival was observed at test item concentrations of 125 μg/ml and above due to toxicity.

RANGE-FINDING/SCREENING STUDIES:
The testing material was tested dose 31.3 to 1000 μg/ml in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix, the relative suspension growth was 4% at the test item concentration of 62.5 μg/ml compared to the relative suspension growth of the solvent control. In the presence of S9-mix, the relative suspension growth was 62% at the test item concentration of 62.5 μg/ml compared to the relative suspension growth of the solvent control.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Mutation frequency per 106 survivors
- S9-mix - S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 857 688 1710
SD 246 187 815
n 110 102 139
Upper control limit(95%) 1425 1124 4214
Lower control limit (95%) 289 253 -793

- Negative historical control data
Mutation frequency per 106 survivors
- S9-mix - S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 86 81 87
SD 23 26 28
n 220 202 273
Upper control limit(95%) 135 135 145
Lower control limit (95%) 37 28 28

SD = Standard deviation, n = Number of observations
Distribution historical negative control data from experiments performed between January 2013 and November 2016.

In the first experiment in the absence of S9-mix, the mutation frequency of one of the solvent control cultures was not within the range of the historical control data. Since the mutation frequency was just above the upper limit of the acceptability criteria range, clear negative results are observed and the mutation frequency of the other solvent control culture was within the historical range, this deviation in the mutation frequency had no effect on the validity of the results of the first mutation experiment. The other values were within the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxiciy: cells were subcultured and counted with coulter particle counter
- Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT).

OTHER INFORMATION ON RESULTS
First mutagenicity test, the dose levels selected to measure mutation frequencies at the TK-locus were:
-Without S9-mix: 5, 10, 20, 30, 40, 50, 60 and 70 μg/ml exposure medium. (The dose level of 80 μg/ml was not used for mutation frequency measurement, this dose level was too toxic for further testing.)
-With S9-mix: 5, 10, 20, 30, 60, 70, 80 and 90 μg/ml exposure medium. (The dose levels of 100 and 110 μg/ml were not used for mutation frequency measurement, since these dose levels showed an inconsistent RSG.)
In the absence of S9-mix (Table 3), the relative total growth of the highest test item concentration was 14% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test item concentration was 15% compared to the total growth of the solvent controls.

Second mutagenicity test, the dose levels selected to measure mutation frequencies at the TK-locus were:
-Without S9-mix with a 24 hour treatment period: 5, 10, 20, 30, 60, 70, 80, 90, 100 and 110 μg/ml (The dose levels of 70 to 110 μg/ml were not used for mutation frequency measurement, these dose levels were too toxic for further testing.) The relative total growth of the highest test item was 37% compared to the total growth of the solvent controls.
- In the second mutagenicity test, an increase above the MF(controls) + 126 (269 per 106 survivors) was observed at the mid dose of 10 μg/ml. Although this increase is above the MF(controls) + 126, no dose-related response is observed and no increases were observed in the first experiment, therefore this increase is considered to be not biologically relevant.

Conclusions:
Under the conditions of this test, Ylang Ylang I was concluded to be negative for the potential to induce forward mutations at the kinase (TK) locus in L5178Y mouse lymphoma cells, in absence and presence of S9-activated test systems. Therefore, the substance does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).
Executive summary:

Ylang Ylang I was tested in accordance with OECD TG 490 for its ability to induce forward mutations at the kinase (TK) locus in L5178Y mouse lymphoma cells, in absence and presence of S9-activated test systems. In the first experiment, Ylang Ylang I was tested up to concentrations of 70 and 90 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 14% and 15% in the absence and presence of S9-mix, respectively. In the second experiment, Ylang Ylang I was tested up to concentrations of 60 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 37%. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Ylang Ylang I did not induce a biologically relevant increase in the mutation frequency in absence or presence of S9-mix. Under the conditions of this test, Ylang Ylang I was found not to induce forward mutations at the kinase (TK) locus in L5178Y mouse lymphoma cells, in absence and presence of S9-activated test systems. Therefore, the substance does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene mutation in bacteria

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 strain specific 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.

Micronucleus in human peripheral blood lymphocytes (HPBL)

Ylang oil I was tested for its potential to induce micronucleus formation in the in vitro micronucleus test with isolated human lymphocytes from fresh whole human blood, according to OECD TG 487. A preliminary toxicity study was performed with dose ranges from 0.5 to 5000 μg/mL. Cytotoxicity (≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) was observed. Based on these results, the doses chosen for the micronucleus assay ranged from 5 to 500 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 1 to 500 μg/mL for the non-activated 24-hour exposure group. The S9 fraction was obtained from Aroclor 1254-induced rat liver. Ethanol was used as a vehicle in the test system. Visible precipitation and hemolysis were observed under all treatment conditions.

Cytokinesis of the cells was blocked using cytochalasin B. Calculation of the Cytokinesis Block Proliferation Index (CBPI; at least 500 cells from appropriate cultures were scored) was used to demonstrate cell division and in order to select doses to process and score microscopically for micronuclei. For the selected doses, the micronucleus frequency was determined from 1000 from each culture (2000 binucleated cells per concentration). The results for positive and negative controls indicate that all criteria for a valid assay were met. No significant or dose-dependent increases in micronuclei induction were observed in the treatment groups with and without S9. Under the conditions of the assay described in this report, Ylang oil I was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Gene mutation in mouse lymphoma cells (MLA)

Ylang Ylang I was tested in accordance with OECD TG 490 for its ability to induce forward mutations at the kinase (TK) locus in L5178Y mouse lymphoma cells, in absence and presence of S9-activated test systems.In the first experiment, Ylang Ylang I was tested up to concentrations of 70 and 90 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 14% and 15% in the absence and presence of S9-mix, respectively. In the second experiment, Ylang Ylang I was tested up to concentrations of 60 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 37%. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Ylang Ylang I did not induce a biologically relevant increase in the mutation frequencyin absence or presence of S9-mix. Under the conditions of this test, Ylang Ylang I was found not to induce forward mutations at the kinase (TK) locus in L5178Y mouse lymphoma cells, in absence and presence of S9-activated test systems.

Justification for classification or non-classification

Based on the available data, Ylang Ylang Ext., I and II does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).