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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:
21-01-2014 until 24-01-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Skin Ethic RHE / S / 17
- Tissue batch number(s): 14 RHE 0101
- Production date: 20 January 2014
- Shipping date: 21 January 2014
- Delivery date: 21 January 2014
- Date of initiation of testing: 21 january 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 mL PBS, 25 times

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: SpectraMax M5 (Molecular Devices)
- Wavelength: 570 nm
- Filter / Filter bandwidth / Linear OD range of spectrophotometer: No data

NUMBER OF REPLICATE TISSUES: Test was performed in triplicate

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean tissue viability after exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the mean tissue viability after exposure is more than 50%.

ACCEPTABIILITY CRITERIA
- The absolute absorbance at 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean absorbance of the three negative controls is ≥ 1.2 and ≤2.5
- An assay is meeting the acceptance criterion if the mean relative tissue viability of the positive control is < 40%.
- The standard deviations (calculated from individual precental tissue viabilities) among tissues of the same treatment group should be ≤18%.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Cell viability at day 17 is 1.475 +/- 0.021 where an O.D. of > 0.7 is accepted
- Barrier function: Exposure time inducing 50% viability (using Triton X-100 1%) is 4.8h where 4-10h is accepted
- Morphology / contamination: The culture and histology of the skin is found to be conform (sufficient cell layers and good cell growth and sterility
- Reproducibility: The system is fully reproducible as it is commercially being sold by SkinEthic.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 16 µL

NEGATIVE CONTROL
- Amount(s) applied: 16µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL
- Concentration: 5% in deionised water
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 ± 1 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
9.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

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

Summary of results

Dose group

Absorbance 570 nm

Mean Absorbance of 3 tissues**

Relative viability [%] Tissue 1, 2, 3**

Relative viability [% of negative control]**

Standard deviation [%]**

Tissue 1*

Tissue 2*

Tissue 3*

Negative control (PBS)

2.091

2.178

2.127

2.132

98.1

100.0

2.0

102.2

99.8

Positive control (5% w/v SDS)

0.013

0.016

0.015

0.015

0.6

0.7

0.1

0.7

0.7

Test substance

0.237

0.152

0.190

0.193

11.1

9.1

2.0

7.2

8.9

*Mean of three replicate wells (per tissue) after blank correction

**Calculation based on exact means

Applicant's summary and conclusion

Interpretation of results:
other: Category 2 (irritant)
Remarks:
based on CLP criteria
Conclusions:
Under the conditions of this test, the relative mean tissue viability for the test item determined to be 9.1%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, it can be concluded that Ethyllinalyl acetate is at least an irritant to skin and further research is needed to be able to assign a classification in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The test substance was tested according to OECD 439 (non-GLP) with the in vitro reconstructed human epidermis model. 16µL test substance was applied and the tissues were exposed for 42 minutes at room temperature, in triplicate. After exposure the tissues were rinsed with PBS. After 42 hours post-incubation a MTT assay was performed to determine the cell viability. Negative and positive controls were run in parallel and acceptance criteria were met. Under the conditions of this test, the relative mean tissue viability for the test item determined to be 9.1%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, it can be concluded that Ethyllinalyl acetate is at least an irritant to skin and further research is needed to be able to assign a classification in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).