Registration Dossier

Administrative data

Description of key information

Skin corrosion (OECD TG 431): not corrosive

Skin irritation (OECD TG 439): irritant (read-across from Ylang Ylang III)

Eye irritation (OECD TG 437): not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-12-2017 to 08-12-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
July 29, 2017
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
May 31, 2008
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Provided by sponsor
- Expiration date of the lot/batch: 30 November 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

OTHER SPECIFICS: UVCB
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek EpiDerm Reconstructed Human Epidermis
Justification for test system used:
A human three dimensional epidermal test, is recommended in international guidelines (e.g. OECD and EC) to minimise the need of in vivo testing.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): EPI-200, Lot no.: 27636 Kit L and Kit M
- Other: The Skin models tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Test item incubation of 3 minutes at room temperature, or 1 hour at 37.0 ± 1.0°C (actual range 36.2 - 37.3°C).
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.2 - 37.3°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT. Since the %NSMTT ≤ 0.0, there is no need to correct for interference of the test item.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

DECISION CRITERIA
- The test substance is considered to be corrosive to skin if
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability equal to or above 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
- The test substance is considered to be non-corrosive to skin if
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

ACCEPTANCE OF RESULT
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be < 30%.
d) The %NSC should be < 30% relative to the negative control OD.
e) The non-specific MTT reduction should be < 30% relative to the negative control OD.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50μL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50μL
- Concentration (if solution): undiluted Milli-Q

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50μL
- Concentration (if solution): 8N KOH
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours (in MTT medium)
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean 3-minute exposure
Value:
109
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean 1-hour exposure
Value:
110
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: Yes, but since the %NSMTT ≤ 0.0, there is no need to correct for interference of the test item
- Colour interference with MTT: Yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test was performed by a GLP laboratory that routinely performes these tests.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the two tissues of the negative control was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The mean relative tissue viability following 1-hour exposure to the positive control was 13% which is below the acceptable percentage of maximum 15%.
- Acceptance criteria met for variability between replicate measurements: In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates was <8% which is below the acceptable percentage of maximum 30%.
Interpretation of results:
other: Not corrosive
Remarks:
based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
Under the conditions of this study, Ylang Ylang I showed a relative mean tissue viability above 100% after the 3-minute and 1-hour treatment. Ylang Ylang I is therefore considered to be not corrosive and therefore does not need to be classified in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin corrosion potential of Ylang Ylang 1 was tested according to OECDTG431. A human three dimensional epidermal model (EpiDerm) was exposed topically to 50 μL undiluted Ylang Ylang 1, Milli-Q (negative control), or Potassium hydroxide (positive control) for 3 minutes or 1 hour. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. Both the negative and the positive control were considered valid. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Ylang Ylang I compared to the negative control tissues was 109% and 110% respectively. Ylang Ylang I is therefore considered to be not corrosive and therefore does not need to be classified in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-04-2017 to 02-06-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 20, 2012
GLP compliance:
yes
Specific details on test material used for the study:
Name of test material as cited in study report: Ylang Ylang III / Test Facility test item 208293 / A
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small Model (EPISKIN-SM, 0.38 cm2, Batch no.: 17-EKIN-016 and 17-EKIN-22), SkinEthic Laboratories, Lyon, France.
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, )
- Tissue batch number(s): Batch no.: 17-EKIN-016 and 17-EKIN-22
- Production date: 30 May 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.2 - 37.7°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Observable damage due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Incubation time: 3 hours at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.

NUMBER OF REPLICATE TISSUES: 3

DECISION CRITERIA
A test item is considered irritant in the skin irritation test if:
-The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
-The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 μL undiluted
NEGATIVE CONTROL
- Amount(s) applied: 25 μL undiluted PBS
POSITIVE CONTROL
- Amount(s) applied: 25 μL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean experiment 1
Value:
43
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: First experiment
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean experiment 2
Value:
63
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Repeat experiment
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 8.8 and 8.1 in the first and second experiment respectively The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 14%, for the negative and positive control indicating that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Since the individual values of the first experiment were both above and below 50% (39, 40 and 51% respectively) the test was inconclusive and a repeat experiment was performed. In the second test, the mean tissue viability was 63% (58, 67 and 65% respectively), which is above the threshold of 50% for irritation.

Interpretation of results:
other: Skin irritant
Remarks:
based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
Under the conditions of this test, the relative mean tissue viability for the test item determined to be respectively 43% and 63%. The value of the first experiment is below the threshold for irritancy of ≤50%, but this experiment was found to be inconclusive. The repeat experiment was negative for irritation. Based on the results obtained, it can be concluded that Ylang Ylang III is worst-case an irritant to skin and should be classified as such in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin irritation potential of Ylang Ylang III was tested in accordance with OECDTG439. Undiluted Ylang Ylang III was topically applied to the skin model for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. Ylang Ylang III did not interact with the MTT. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Ylang Ylang III compared to the negative control tissues was 43%. Since the individual values were both above and below 50% (39, 40 and 51% respectively) the test was inconclusive and a repeat experiment was performed. In the second test, the mean tissue viability was 63% (58, 67 and 65% respectively). Both the positive and the negative control were within the historical control data range and therefore considered valid. Furthermore, the standard deviation value of the percentage viability of three tissues treated identically was less than 14%, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item determined to be respectively 43% and 63%. The value of the first experiment is below the threshold for irritancy of ≤50%, but this experiment was found to be inconclusive. The repeat experiment was negative for irritation. Based on the results obtained, it can be concluded that Ylang Ylang III is worst-case an irritant to skin and could be classified as such in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The full read across justification report is attached under "Attached justification":
05 February 2018 READ-ACROSS STUDY / YLANG YLANG EXT /I /II OIL -SKIN IRRITATION I&B9W8768R001F0.2\

Executive Summary
According to Annex VII, 8.1 of the REACh Regulation (EC) No 1907/2006, skin irritation is standard information required for the registration of substances manufactured or imported in quantities of one tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

A read-across approach appears as appropriated to predict the endpoint “Skin irritation” for the target substance Ylang Ylang Ext /I /II oil because:
Ylang Ylang Ext /I /II oil and Ylang Ylang III oil, are very similar in composition and show mainly variations in concentrations of constituents. A large number of these constituents are notified for skin irritation, and the QSAR toolbox gives various alerts for reactive structures among the constituents.
A skin irritation study according to OECD test guideline 439, is available for the substance Ylang Ylang III oil.
The source substance Ylang Ylang III oil has been classified for skin irritation, and based on the composition similarity regarding reactive constituents of the source and target substance, the results for skin irritation can be read across to the target substance.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the skin irritation potential for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter.
Reason / purpose:
read-across source
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean experiment 1
Value:
43
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: First experiment
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean experiment 2
Value:
63
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Repeat experiment
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 8.8 and 8.1 in the first and second experiment respectively The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 14%, for the negative and positive control indicating that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Since the individual values of the first experiment were both above and below 50% (39, 40 and 51% respectively) the test was inconclusive and a repeat experiment was performed. In the second test, the mean tissue viability was 63% (58, 67 and 65% respectively), which is above the threshold of 50% for irritation.

Interpretation of results:
other: Skin irritant
Remarks:
based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
Under the conditions of this test, the relative mean tissue viability for the test item determined to be respectively 43% and 63%. The value of the first experiment is below the threshold for irritancy of ≤50%, but this experiment was found to be inconclusive. The repeat experiment was negative for irritation. These results are read across to the target substance Ylang Ylang Ext/I / II oil. Based on the results obtained, it can be concluded that this substance is an irritant to skin (worst-case) and should be classified as such in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin irritation potential of Ylang Ylang III was tested in accordance with OECDTG439. Undiluted Ylang Ylang III was topically applied to the skin model for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. Ylang Ylang III did not interact with the MTT. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Ylang Ylang III compared to the negative control tissues was 43%. Since the individual values were both above and below 50% (39, 40 and 51% respectively) the test was inconclusive and a repeat experiment was performed. In the second test, the mean tissue viability was 63% (58, 67 and 65% respectively). Both the positive and the negative control were within the historical control data range and therefore considered valid. Furthermore, the standard deviation value of the percentage viability of three tissues treated identically was less than 14%, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item determined to be respectively 43% and 63%. The value of the first experiment is below the threshold for irritancy of ≤50%, but this experiment was found to be inconclusive. The repeat experiment was negative for irritation. These results are read across to the target substance Ylang Ylang Ext/I / II oil. Based on the results obtained, it is concluded that the substance is an irritant to skin (worst-case) and could be classified as such in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-04-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
July 26, 2013
GLP compliance:
yes
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: 09 August 2018
- Purity test date: 09 August 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Vitelco slaughterhouse, 's Hertogenbosch, The Netherlands
- Characteristics of donor animals (e.g. age, sex, weight): Young cattle
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Indication of any existing defects or lesions in ocular tissue samples: Those corneas exhibiting defects were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μl of either the negative control, positive control or undiluted test item was introduced onto the epithelium of the cornea.
Duration of treatment / exposure:
10 +/- 1 minutes
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
-The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum ). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS:
- Opacity determinations were performed on each of the corneas using an opacitometer (BASFOP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes, physiological saline

POSITIVE CONTROL USED: Yes, ethanol

APPLICATION DOSE AND EXPOSURE TIME: Test item applied undiluted for 10 +/- 1 minutes at 32 +/- 1°C.

TREATMENT METHOD: The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2

POST-EXPOSURE INCUBATION: Yes, 120 +/- 10 minutes at 32 +/- 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microplate reader (TECAN Infinite® M200 Pro Plate Reader).

SCORING SYSTEM: The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
1) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
2) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range. All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

EVALUATION CRITERIA
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS
Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN
GHS No Category) are:
- ≤3: No Category
- >3 ≤ 55: No prediction can be made
- >55: Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
Main experiment
Value:
1.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 61 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline:
Negative control
- Opacity: 2.9-3.0 (SD 1.07, n=72)
- Permeability: 0.016-0.042 (SD 0.01, n=65)
- IVIS: 2.8-3.0 (SD 1.17, n=66)
Positive control:
- IVIS: 34.7-78.2 (SD 12.64, n=47)

Summary of Opacity, Permeability and In Vitro Scores

Treatment  Mean
Opacity (1)
Mean
Permeability (1)
Mean In vitro
Irritation Score (1,2)
Negative control 0.1 0.005 0.1
Positive control
(Ethanol)
19.0 1.992 48.9
Ylang Ylang I  1.6 0.003 1.6

(1) Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

(2) In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Interpretation of results:
other: Not classified
Remarks:
based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
Under the conditions of this test, an IVIS of 1.6 was found for Ylang Ylang I. Based on these results, the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).
Executive summary:

The eye hazard potential of Ylang Ylang I was evaluated according to OECD guideline 437 (BCOP test). The eye damage was assessed through topical application of 750 μl of the undiluted Ylang Ylang I for 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Ylang Ylang I did not induce ocular irritation (no opacity and no permeability), given the result of a mean in vitro irritancy score of 1.6 after 10 minutes of treatment which is below the threshold for irritation of 3. Based on this result, the test substance does not need to be classified as eye irritant in accordance with the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin corrosion in vitro

The skin corrosion potential of Ylang Ylang 1 was tested according to OECDTG431. A human three dimensional epidermal model (EpiDerm) was exposed topically to 50 μL undiluted Ylang Ylang 1, Milli-Q (negative control), or Potassium hydroxide (positive control) for 3 minutes or 1 hour. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. Both the negative and the positive control were considered valid. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Ylang Ylang I compared to the negative control tissues was 109%and 110% respectively. Ylang Ylang I is therefore considered to be not corrosive.

Skin irritation in vitro (read-across from Ylang Ylang III)

The skin irritation potential of Ylang Ylang III was tested in accordance with OECD TG 439. Undiluted Ylang Ylang III was topically applied to the skin model for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. Ylang Ylang III did not interact with the MTT. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Ylang Ylang III compared to the negative control tissues was 43%. Since the individual values were both above and below 50% (39, 40 and 51% respectively) the test was inconclusive and a repeat experiment was performed. In the second test, the mean tissue viability was 63% (58, 67 and 65% respectively). Both the positive and the negative control were within the historical control data range and therefore considered valid. Furthermore, the standard deviation value of the percentage viability of three tissues treated identically was less than 14%, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item determined to be respectively 43% and 63%. The value of the first experiment is below the threshold for irritancy of ≤50%, but this experiment was found to be inconclusive. The repeat experiment was negative for irritation. Based on the results obtained, it can be concluded that Ylang Ylang III is worst-case an irritant to skin.

Eye irritation in vitro

The eye hazard potential of Ylang Ylang I was evaluated according to OECD TG 437 (BCOP test). The eye damage was assessed through topical application of 750 μl of the undiluted Ylang Ylang I for 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Ylang Ylang I did not induce ocular irritation (no opacity and no permeability), given the result of a mean in vitro irritancy score of 1.6 after 10 minutes of treatment which is below the threshold for irritation of 3. Based on this result, the test substance does not need to be classified as eye irritant in accordance with the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Justification for classification or non-classification

Based on the available data, Ylang Ylang Ext., I and II needs to be classified as skin irritant (H315 / Skin Irrit. 2) but not as corrosive or eye irritant in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).