Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Pale yellow clear liquid
Details on test material:
Name of test material as cited in study report: Ylang Ylang I / Test Facility test item 208028 / B
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
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

In vitro test system

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

Results and discussion

In vitro

Resultsopen allclose all
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%.

Applicant's summary and conclusion

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).