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Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD TG 471): negative

In vitro cytogenicity / micronucleus study (OECD TG 487): negative

In vitro gene mutation study 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:
31-03-2017 to 23-04-2017
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
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 Jan 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not provided by sponsor

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: 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate
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:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
precipitation was observed beginning at 1500 μg
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
precipitation was observed under all conditions beginning at 1500 μg
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- First mutation experiment: Precipitate was observed beginning at 1500 μg per plate with all conditions. Toxicity was observed beginning at 1500 or at 5000 μg per plate with a few conditions.
- Second mutation experiment: Precipitate was observed beginning at 1500 μg per plate with all conditions. Toxicity was observed beginning at 1500 or at 5000 μg per plate with tester strain TA100 in the presence and absence of S9 activation.

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:
Based on the results of this study it is concluded that Ylang Oil III 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 III was evaluated according to guideline OECD TG 471. Ylang Oil III was tested in Salmonella typhimurium tester strains TA1535, TA1537, TA98, TA100 and Escherichia coli tester strain WP2uvrA 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: 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate. Precipitation of Ylang Oil III on the plates was observed from 1500 μg/plate with all conditions. Cytotoxicity, as evidenced by a decrease in the number of revertants, was not observed in the first experiment beginning at 1500 or at 5000 μg per plate with a few conditions. Ylang Oil III 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 WP2uvrA, 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. Based on the results of this study it is concluded that Ylang Oil III 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 30-05-2017
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)
Version / remarks:
2014
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 and lot/batch No.of test material: Obtained from sponsor, RIFM # 6614-F2.12.3
- Expiration date of the lot/batch: 26 Jan 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Solubility and stability of the test substance in the solvent/vehicle: 500 mg/mL in ethanol
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: compatible

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test substance dilutions were prepared in ethanol immediately before use and delivered to the test system at room temperature under filtered light.
- Final dilutions of a liquid or gel: 1, 5, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 µg/mL;

Species / strain / cell type:
lymphocytes: Human Peripheral Blood Lymphocytes (HPBL)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healthy non-smoking human. The donor had no recent history of radiotherapy, viral infection or the administration of drugs and who had abstained from alcohol for 12 or more hours prior to blood donation.
- Suitability of cells: This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals (Clare et al., 2006).
- Sex, age and number of blood donors if applicable: one female 26 years of age at 1st collection and 27 years at 2nd collection.
- Whether whole blood or separated lymphocytes were used if applicable: separated lymphocytes
- Number of passages if applicable: 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 (Fenech and Morley, 1986).
- Methods for maintenance in cell culture if applicable: The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI-1640 containing 15% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin. 5 ± 1% CO2 in air
- Properly maintained: Yes
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Cytochalasin B (cytoB) dissolved in DMSO (stock concentration of 2 mg/mL, dissolved to 6µg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Cytotoxicity was observed at doses ≥ 50 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 150 μg/mL in the S9-activated 4-hour exposure group. Based upon the results of the preliminary toxicity assay, the doses were selected for the micronucleus assay.

Non-activated (4hr) 5, 15, 20, 25, 30, 35, 40, 45, 50, 100 µg/mL
Non-activated (24 hr) 1, 5, 15, 20, 25, 30, 35, 40, 45, 50 µg/mL
S9-activated (4 hr) 5, 25, 50, 60, 70, 80, 90, 100, 125, 150 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: based on the solubility of the test substance and compatibility with the target cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Vinblastine (VB)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hr and 24 hr
- Expression time (cells in growth medium): 20 hr
- Fixation time (start of exposure up to fixation or harvest of cells): overnight

STAIN (for cytogenetic assays): acridine orange

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and stored overnight or longer at 2-8°C. To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried before staining.

NUMBER OF CELLS EVALUATED: 1000

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
Rationale for test conditions:
Vehicle Controls: The frequency of cells with micronuclei should be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are not extreme outliers (indicative of experimental or human error).
Positive Controls: The percentage of micronucleated cells must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).
Cell Proliferation: The CBPI of the vehicle control at harvest must be ≥ 1.4.
Test Conditions: The test substance must be tested using a 4-hour treatment with and without S9, as well as a 24-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.
Analyzable Concentrations: At least 2000 binucleated cells from at least three appropriate test substance concentrations.
Evaluation criteria:
The test substance was considered positive when:
• 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:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 45 μg/mL (4h) 35 μg/mL (24h)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: Human Peripheral Blood Lymphocytes
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 90 μg/mL (4h)
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 effects
- Effects of osmolality: no effects
- Precipitation: >150 µg/mL at the beginning and end of the treatment
- Other confounding effects: hemolysis ≥ 500 μg/mL

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity was observed at doses ≥ 50 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 150 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 150 μg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 100 μg/mL for the non-activated 4-hour exposure group; from 5 to 150 μg/mL for the S9-activated 4-hour exposure group; and from 1 to 50 μg/mL for the non-activated 24-hour exposure group. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 100 μg/mL for the non-activated 4-hour exposure group; from 5 to 150 μg/mL for the S9-activated 4-hour exposure group; and from 1 to 50 μg/mL for the non-activated 24-hour exposure group.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See table

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: See table
- Indication whether binucleate or mononucleate where appropriate: See table

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

MICRONUCLEUS ASSAY USING Ylang Oil III

 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 24-HOUR HARVEST

Treatment Condition Replicate Culture Total # of Cells Count per total cells Count per total cells Count per total cells CBPI Cytotoxicity
   Cells with # of nuclei      Cells with # of nuclei      Cells with # of nuclei  
µg/mL   Counted 1 2 >2    
Ethanol A 500 150 329 21 1.733
B 500 157 324 19
Ylang Oil III
5 A 500 153 324 23 1.737 -1%
B 500 154 325 21
15 A 500 165 322 13 1.701 4%
B 500 159 329 12
20 A 500 183 299 18 1.681 7%
B 500 163 328 9
25 A 500 169 327 4 1.669 9%
B 500 172 322 6
30 A 500 182 316 2 1.666 9%
B 500 161 332 7
35 A 500 164 331 5 1.673 8%
B 500 172 324 4
40 A 500 206 292 2 1.604 18%
B 500 193 306 1
45 A 500 307 193 0 1.338 54%
B 500 355 145 0
50 A 500 402 98 0 1.163 78%
B 500 435 65 0
100 A 0 0 0 0 0.000 -
B 0 0 0 0

IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 24-HOUR HARVEST

Treatment Condition Replicate Culture Total # of Cells Count per total cells Count per total cells Count per total cells CBPI Cytotoxicity
   Cells with # of nuclei      Cells with # of nuclei      Cells with # of nuclei  
µg/mL   Counted 1 2 >2    
Ethanol A 500 162 327 11 1.693  
B 500 168 320 12
Ylang Oil III
5 A 500 178 314 8 1.675 3%
B 500 165 325 10
25 A 500 179 320 1 1.657 5%
B 500 172 321 7
50 A 500 183 317 0 1.633 9%
B 500 189 306 5
60 A 500 184 314 2 1.644 7%
B 500 175 324 1
70 A 500 210 285 5 1.618 11%
B 500 179 319 2
80 A 500 308 192 0 1.371 46%
B 500 323 175 2
90 A 500 365 135 0 1.302 56%
B 500 333 167 0
100 A 500 370 130 0 1.215 69%
B 500 415 85 0
125 A 500 462 38 0 1.068 90%
B 500 471 28 1
150 A 0 0 0 0 0.000 -
B 0 0 0 0
CP, 2.5 A 500 193 307 0 1.628 9%
B 500 179 321 0
CP, 5 A 500 222 278 0 1.516 26%
B 500 262 238 0
CP, 7.5 A 500 372 128 0 1.325 53%
  B 500 303 197 0    

IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 24-HOUR TREATMENT, 24-HOUR HARVEST

Treatment Condition Replicate Culture Total # of Cells Count per total cells Count per total cells Count per total cells CBPI Cytotoxicity
   Cells with # of nuclei      Cells with # of nuclei      Cells with # of nuclei  
µg/mL   Counted 1 2 >2    
Ethanol A 500 162 304 34 1.728  
B 500 175 294 31
Ylang Oil III
1 A 500 169 311 20 1.700 4%
B 500 179 293 28
5 A 500 185 296 19 1.685 6%
B 500 173 303 24
15 A 500 187 297 16 1.654 10%
B 500 194 287 19
20 A 500 224 266 10 1.582 20%
B 500 217 270 13
25 A 500 232 252 16 1.575 21%
B 500 223 263 14
30 A 500 239 257 4 1.522 28%
B 500 244 255 1
35 A 500 345 155 0 1.354 51%
B 500 301 199 0
40 A 500 406 94 0 1.181 75%
B 500 413 87 0
45 A 500 475 25 0 1.083 89%
B 500 442 58 0
50 A 0 0 0 0 0.000 -
B 0 0 0 0
VB, 5 mg/mL A 500 186 276 38 1.743 -2%
B 500 157 295 48
VB, 7.5 ng/mL A 500 174 285 41 1.684 6%
B 500 195 293 12
VB, 10 ng/mL A 500 272 223 5 1.453 38%
  B 500 289 202 9    
Conclusions:
Based on the results of this study it is concluded that Ylang Ylang Oil III 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 test substance, Ylang Ylang Oil III, was tested for the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in accordance with OECD Guideline 487, under GLP conditions. Target cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Ethanol was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 μg/mL. Based on these results, the doses chosen for the micronucleus assay ranged from 5 to 100 μg/mL for the non-activated 4-hour exposure group; from 5 to 150 μg/mL for the S9-activated 4-hour exposure group; and from 1 to 50 μg/mL for the non-activated 24-hour exposure group.In the micronucleus assay, cytotoxicity was observed at doses ≥ 45 μg/mL in the 4-hour exposure group without S9; at doses ≥ 90 μg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 35 μg/mL in the 24-hour exposure group without S9.

The doses selected for evaluation of micronuclei were:

-       15, 30, and 45 μg/mL for the non-activated 4-hour exposure group;

-       25, 60, and 90 μg/mL for the S9-activated 4-hour exposure group; and

-       5, 20, and 35 μg/mL for the non-activated 24-hour exposure group.

No significant or dose-dependent increases in micronuclei induction were observed in treatment groups with or without S9. These results indicate Ylang Ylang Oil III was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Aug 2017 - 19 Feb 2018
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)
Version / remarks:
2016
Deviations:
yes
Remarks:
Due to the changed appearance of the test item, another batch of the test item was used for the additional experiment.
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Two batches of the same material were used for this study, as the appearance of the available test sample at the CRO became turbid over time.
Target gene:
thymidine kinase (TK)
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: Recommended test system in international guidelines (e.g. OECD, EC).
- Density: Cell density will be preferably kept below 1 x 10^6 cells/ml.
- Whether whole blood or separated lymphocytes were used if applicable: lymphocytes
- Methods for maintenance in cell culture if applicable:

All incubations will be carried out in a humid atmosphere (80 - 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. Temperature and humidity will be continuously monitored throughout the experiment. The CO2 percentage will be monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions will be evaluated and maintained in the raw data.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2mM L-glutamin. 5.0 ± 0.5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the results of the dose-range finding test, the following dose-range was selected for the mutagenicity test:
Without S9-mix: 0.47, 0.94, 1.88, 3.75, 7.5, 15, 20, 25, 30, 35, 40 and 50 μg/mL exposure medium.
With S9-mix: 1.88, 3.75, 7.5, 15, 30, 60, 75, 90, 115, 130, 145 and 160 μg/mL exposure medium.

In practice (based on cytotoxicity observed) the below concentrations were used in the final experiments
Experiment 1
Without S9-mix: 1.25, 2.5, 5, 10, 20, 25, 30 and 40 μg/mL exposure medium.
With S9-mix: 3.75, 7.5, 15, 30, 60, 75, 90 and 115 μg/mL exposure medium.

Experiment 2
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 7.5, 15, 22.5, 30, 35, 40, 45, 50, 55, 60, 70 and 80 µg/mL exposure medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Homogeneity was observed (visible) for the test substance using this commonly used vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Remarks:
no remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 8 x 10^6 cells (10^6 c/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 c/mLfor 24 hour treatment)

DURATION
- Exposure duration: 3h or 24h
- Expression time (cells in growth medium): 2 days after the start of the treatment period
- Selection time (if incubation with a selection agent): 11 or 12 days
- Staining time: 2 hours (incubation with MTT)

SELECTION AGENT (mutation assays): TFT-selection

NUMBER OF REPLICATIONS: two separate experiments, the solvent control was tested in duplicate.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
In accordance with OECD TG 490
Evaluation criteria:
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 x 10^-6 .
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. A Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction (ToxRat Professional v 3.2.1). Any 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.

In addition: any increase of the mutation frequency is evaluated for its biological relevance including a comparison of the results with the historical control data range.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
other: The mean mutation frequency 1st experiment: within the range of the acceptability criteria The mean mutation frequency 2nd experiment: one of the solvent control cultures was not within the range of the acceptability criteria.
Remarks:
Validity justification: Since the mutation frequency was just above the higher limit of the acceptability criteria range and the mutation frequency of the other solvent control culture was within the acceptability criteria range.
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Limited solubility
- Precipitation: The 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 626 μg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
3h exposure: In the absence of S9-mix, the relative suspension growth was 11% at the test item concentration of 31.3 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 62.5 μg/mL and above.In the presence of S9-mix, the relative suspension growth was 66% at the test item concentration of 62.5 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test item concentrations of 125 μg/mL and above.
24 hours exposure: The relative suspension growth was 67% at the test item concentration of 31.3 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentration of 62.5 μg/mL and above.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) - frequency per 10^6 survivors
3 hour
- Without S9: Mean 808, SD 239, 95% CI (152 - 1465)
- With S9: Mean 1669, SD 843, 95% CI (-859 - 4196)
24 hour
- Without S9: Mean 684, SD 206, 95% CI (146 - 1222)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Other observations when applicable: Ylang Ylang III was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 1000 μg/mL.

Using the dose ranges based on the dose-range test no appropriate levels of toxicity were observed in the first mutagenicity test, and therefore the experiment was repeated (repeat test 1A-1D) and the below doses were used as final dose levels in experiment 1:

Experiment 1

Without S9-mix: 1.25, 2.5, 5, 10, 20, 25, 30 and 40 μg/mL exposure medium.

With S9-mix: 3.75, 7.5, 15, 30, 60, 75, 90 and 115 μg/mL exposure medium.

In the absence of S9-mix the relative total growth of the highest test item concentration was 15% 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 16% compared to the total growth of the solvent controls.

Experiment 2

Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 

7.5, 15, 22.5, 30, 35, 40, 45, 50, 55, 60, 70 and 80 µg/mL exposure medium.

The dose levels of 30 and 50 to 80 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing or showed an inconsistent RSG (30 µg/mL). 

The dose levels selected to measure mutation frequencies at the TK-locus were: 7.5, 15, 22.5, 35, 40 and 45 µg/mL exposure medium.

The relative total growth of the highest test item was 12% compared to the total growth of the solvent controls

Conclusions:
Under the conditions of this test, Ylang Ylang III 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 III 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 III was tested up to concentrations of 40 µg/mL and 115 µg/mL in the absence (batch 17066411) and presence (batch 44858) S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 15% and 16% in the absence and presence of S9-mix, respectively. In the second experiment, Ylang Ylang III (batch 44858) was tested up to concentrations of 45 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 12%. 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. In either the presence or the absence of S9-mix, Ylang Ylang III did not induce a significant increase in the mutation frequency at the kinase (TK) locus in L5178Y mouse lymphoma cells. 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

Genetic toxicity in vitro (Ames)

The mutagenic potential of Ylang Oil III was evaluated according to guideline OECD TG 471. Ylang Oil III was tested in Salmonella typhimurium tester strains TA1535, TA1537, TA98, TA100 and Escherichia coli tester strain WP2uvrA 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: 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate. Precipitation of Ylang Oil III on the plates was observed from 1500 μg/plate with all conditions. Cytotoxicity, as evidenced by a decrease in the number of revertants, was not observed in the first experiment beginning at 1500 or at 5000 μg per plate with a few conditions. Ylang Oil III 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 WP2uvrA, 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. Based on the results of this study it is concluded that Ylang Oil III is not mutagenic.

 

In vitro cytogenicity / micronucleus study

The test substance, Ylang Ylang Oil III, was tested for the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in accordance with OECD Guideline 487, under GLP conditions. Target cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Ethanol was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 μg/mL. Based on these results, the doses chosen for the micronucleus assay ranged from 5 to 100 μg/mL for the non-activated 4-hour exposure group; from 5 to 150 μg/mL for the S9-activated 4-hour exposure group; and from 1 to 50 μg/mL for the non-activated 24-hour exposure group.In the micronucleus assay, cytotoxicity was observed at doses ≥ 45 μg/mL in the 4-hour exposure group without S9; at doses ≥ 90 μg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 35 μg/mL in the 24-hour exposure group without S9.

The doses selected for evaluation of micronuclei were: 15, 30, and 45 μg/mL for the non-activated 4-hour exposure group; 25, 60, and 90 μg/mL for the S9-activated 4-hour exposure group; and  5, 20, and 35 μg/mL for the non-activated 24-hour exposure group. No significant or dose-dependent increases in micronuclei induction were observed in treatment groups with or without S9.These results indicate Ylang Ylang Oil III was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

In vitro gene mutation study in mammalian cells

Ylang Ylang III 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 III was tested up to concentrations of 40 µg/mL and 115 µg/mL in the absence (batch 17066411) and presence (batch 44858) S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 15% and 16% in the absence and presence of S9-mix, respectively. In the second experiment, Ylang Ylang III (batch 44858) was tested up to concentrations of 45 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 12%. 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. In either the presence or the absence of S9-mix, Ylang Ylang III did not induce a significant increase in the mutation frequency at the kinase (TK) locus in L5178Y mouse lymphoma cells. 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).

Justification for classification or non-classification

Based on the available information, Ylang Ylang III oil does not need to be classified for genetic toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).