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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
dopted 29 July 2016
Deviations:
not specified
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Remarks:
light yellow
Details on test material:
CH03216 / E1A

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EpiDerm Skin Model (EPI-200)
- Tissue batch number(s):26490 Kit L and K
- Shipping date: 12 July 2017
- Date of initiation of testing: One day after receipt

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out at 37.0 ± 1.0°C (actual range 36.8 – 37.5°C).


REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 2
- Observable damage in the tissue due to washing: /
- Modifications to validated SOP: /

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). Both supplied by MatTek Corporation.
- Incubation time: 3 hours
- Spectrophotometer: The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No interference with MTT

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%. In addition, a test item considered non-corrosive (viability  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 the relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%. In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
The skin was moistened with 25 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 27.7 to 36.7 mg of the solid test item was added into the 6-well plates on top of the skin tissues.

NEGATIVE CONTROL / POSITIVE CONTROL
2 tissues were treated with 50 μl Milli-Q water (negative control) and 2 tissues were treated with 50 μl 8N KOH (positive control)
Duration of treatment / exposure:
The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
105
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <=2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 11%.
In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was  22%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, 3,4,5-TRIMETHOXYCINNAMIC ACID is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate 3,4,5-TRIMETHOXYCINNAMIC ACID for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD (OECD 431, adopted 29 July 2016) and EC (440/2008) guidelines.

Batch CH03216/E1A of the test item was a light yellow crystalline solid with a purity of approximately 99%. Skin tissue was moistened with 25 μl of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 11% after the 1-hour exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptancelimit <=2.8) and the laboratory historical control data range. In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was <=22%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 100% and 105%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.