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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:
The study was conducted between 23 May 2018 and 25 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 18/07/2017 - 20/07/2017 Date of Issue: 28/11/2017

Test material

Constituent 1
Chemical structure
Reference substance name:
trans-cinnamic acid
EC Number:
205-398-1
EC Name:
trans-cinnamic acid
Cas Number:
140-10-3
Molecular formula:
C9H8O2
IUPAC Name:
3-phenylacrylic acid
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20171106
- Expiration date of the lot/batch: 10 April 2020


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark, over silica gel

In vitro test system

Test system:
human skin model
Source species:
other: reconstructed human epidermis
Cell type:
other: normal, human-derived epidermal keratinocytes (NHEK)
Cell source:
other: reconstructed human epidermis
Source strain:
other: reconstructed human epidermis
Details on animal used as source of test system:
Not applicable

Justification for test system used:
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 22 May 2018
EpiDermTM Tissues (0.63cm2) lot number : 28615
Assay Medium lot number : 051718TMA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
2 mL of MTT extractant (isopropanol) was used
to completely immerse each insert and the plate was covered with plate sealer to prevent
Isopropanol evaporation.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Inspected by visual observation
Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
24-well plates; one run

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD570 of test item/mean OD570 of negative control) x 100

Classification of corrosivity potential is based on the relative viabilities for both exposure times according Table 1 below.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
25 mg of test item
Duration of treatment / exposure:
60 minutes/3 minutes
Duration of post-treatment incubation (if applicable):
Overnight
Number of replicates:
24-well plates

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean 3 minute exposure period
Value:
96.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean 60 minute
Value:
94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

Assessment of Colour Interference with the MTT endpoint

The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Test Item, Positive Control Item and Negative Control Item

Mean OD570 values and viabilities for the negative control, positive control and test item are given in the below table:

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

Exposure Period

Mean OD570 of individual tissues

Mean OD570 of duplicate tissues

Standard Deviation

Coefficient of Variation (%)

Relative Mean Viability (%)

 

Negative Control

3 Minutes

2.145

2.217

0.101

4.6

100*

2.288

60 Minutes

1.871

1.922

0.071

3.7

1.972

 

Positive Control

3 Minutes

0.055

0.051

0.006

n/a

2.3

0.047

60 Minutes

0.030

0.040

0.014

n/a

2.1

0.050

 

Test Item

3 Minutes

2.115

2.132

0.023

1.1

96.1

2.148

60 Minutes

1.766

1.806

0.057

3.1

94.1

1.846

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minutes

100*

2.3

96.1

60 minutes

100*

2.1

94.0

*The mean viability of the negative control tissues is set at 100%

Quality Criteria

The mean OD570 for the negative control treated tissues was 2.217 for the 3-Minute exposure period and 1.922 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 2.1% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the

corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for

MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minutes

100*

2.3

96.1

60 minutes

100*

2.1

94.0

* The mean viability of the negative control tissues is set at 100%

Quality Criteria

The quality criteria required for acceptance of the results in the test were satisfied

Conclusion

The test item was considered to be non-corrosive to the skin