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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:
31.08.2016 - 09.09.2016
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
Justification for test system used:
Dermal irritation is generally defined as "the production of reversible inflammatory changes in the skin". The potential for chemical induced skin irritation is usually determined in vivo in the Draize rabbit skin irritation test as described in OECD guideline 404. However, because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDermTM and EpiSkinTM and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SITKit
- Tissue batch number(s): 23353
- Delivery date: 06.09.2016
- Date of initiation of testing: 06.09.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 °C and 25 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at least 15 times
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 42 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Filter: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The substance is considered skin irritant category 2 according to UN GHS (published 2003, last (6th) revision 2015) if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

ACCEPTANCE CRITERIA
- Negative control: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8.
- Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
- Standard deviation: The rel. SD of 3 identical replicates should be < 18%. OD values should not be below historically established boundaries.
- Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system / test kit.
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 tissue runs
Value:
6.4
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
The acceptance criteria were met.

Any other information on results incl. tables

Results

 

Dose Group

Tissue No.

Absorbance 570 nm Well 1

Absorbance 570 nm Well 2

Absorbance 570 nm Well 3

Mean Absorbance of 3 Wells

Mean Absorbance
 of 3 wells blank corrected

Mean Absorbance
of 3 tissues after blank correction *

Rel. Absorbance [%] Tissue 1, 2 + 3**

Relative Standard Deviation [%]

Mean Rel. Absorbance [% of Negative Control]***

Blank

 

0.038

0.038

0.037

0.038

0.000

 

 

 

 

Negative Control

1

2.064

2.078

2.207

2.116

2.079

1.927

107.9

8.2

100.0

2

1.955

2.003

1.978

1.979

1.941

100.7

3

1.768

1.784

1.849

1.800

1.762

91.4

Positive Control

1

0.119

0.122

0.112

0.117

0.080

0.084

4.1

5.2

4.4

2

0.126

0.125

0.128

0.126

0.088

4.6

3

0.129

0.118

0.120

0.122

0.084

4.4

Test Item

1

0.159

0.161

0.164

0.161

0.123

0.124

6.4

1.2

6.4

2

0.165

0.168

0.158

0.164

0.126

6.5

3

0.160

0.160

0.163

0.161

0.123

6.4

* Mean of three replicate wells after blank correction

** Relative absorbance per tissue [rounded values]: 100 × (absorbancetissue) / (mean absorbancenegative control)

*** Relative absorbance per treatment group [rounded values]: 100×(mean absorbancetest item / positive control) / (mean absorbancenegative control)

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test item is to be considered as irritant to skin.
Executive summary:

In this in vitro study the irritation potential of the test item was assessed by means of the Human Skin Model Test. The study was according to OECD 439 and GLP.

The test item did not reduce MTT (test for direct MTT reduction), and did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive (colourless to yellow). Consequently, additional tests with freeze-killed or viable tissues were not necessary.

The main study consists of topical exposure of the test item to the human reconstructed epidermis model followed by a cell viability test. The cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured (as optical density (OD)) after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential and used for the purpose of classification as irritating or non-irritating. The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is ≤ 50%.

Three tissues of the human skin model EpiDermTM were treated with the test item, the negative (DPBS) or the positive (5% SLS) control for 60 minutes.

Hereafter the skin tissues are washed and further incubated for about 42 hours, whereafter the tissues were treated with MTT for 3 hours following about 2.7 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD≥0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease to 4.4% in the relative absorbance as compared to the negative control for the 60 minutes treatement interval, thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 9% (threshold of the OECD TG: < 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value decreased to 6.4% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50% and therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is to be considered as irritant to skin.