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Toxicological information

Skin irritation / corrosion

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Administrative data

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

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
pale yellow to clear yellow
Details on test material:
Name of test material as cited in study report: Ylang Ylang III oil / Test Facility AkzoNobel test item T17032

Name of test material as cited in study report: Ylang Ylang III / Test Facility test item 208293 / A
SOURCE OF TEST MATERIAL
- Source of test material: Obtained from sponsor
- Expiration date of the lot/batch: 31 August 2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stable under storage conditions


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

In vitro test system

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

Results and discussion

In vitro

Resultsopen allclose all
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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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 could 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).