Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Apr - 6 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 Jul 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008, 1st ATP 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
Hex-3-en-1-yl acetate
EC Number:
911-593-9
Molecular formula:
C8H14O2
IUPAC Name:
Hex-3-en-1-yl acetate

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ (EPI-200-SIT)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SIT) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23339
- Delivery date: 31 May 2016
- Date of initiation of testing: 31 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile bench

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.57 ± 0.057 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.51 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT in a pre-test, an additional test with freeze-killed tissues was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is ≤ 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% aqueous solultion
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
approximately 43 h
Number of replicates:
triplicates for each treatment and control group; from each tissue, 3 wells were prepared for absorbance measurements after MTT incubation

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
60 minutes exposure
Value:
67.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
: OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not interfere with the MTT assay (no reducing capacity).
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values (1.525, 1.957 and 1.950) were in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability values of the test substance and the negative control in the main test were 19% (threshold of OECD TG 439: < 18%). The slight excess of the recommended standard deviation by the positive control (19.0%) was still considered acceptable since the positive control produced a distinct positive result with very low absorbance values, and the mean OD values were within the range of historical control data.

Any other information on results incl. tables

Table 2. Results after treatment with the test substance and controls

Test group

Mean absorbance at 570 nm*

Rel. absorbance (%)**

SD (%)

Rel. absorbance (% of negative control)***

Tissue 1

Tissue 2

Tissue 3

Tissue 1

Tissue 2

Tissue 3

Negative control

1.525

1.957

1.950

84.2

108.1

107.7

13.7

100.0

Positive control

0.061

0.082

0.089

3.4

4.6

4.9

19.0

4.3

Test substance

1.453

1.034

1.183

80.2

57.1

65.3

17.3

67.6

* Mean of three replicate wells after blank correction (blank = 0.036)

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test substance/positive control) / (mean absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the reconstructed human epidermis test the test substance does not possess any skin irritating potential.
Executive summary:

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2016). After treatment with the test substance for 60 min the tissue viability decreased to 67.6% compared to the negative control (threshold for irritancy ≤ 50%). Therefore, the test substance is not considered to be irritating to the skin.

Categories Display