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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:
08.08.2016 - 15.08.2016
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden

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-SIT Kit
- Tissue batch number(s): 23349

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

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 positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): undiluted; 47 µL/cm2

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:
1
Value:
99.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no 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 lead 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

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

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

 

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

0.039

0.039

0.038

0.000

 

 

 

 

Negative Control

1

1.658

1.633

1.664

1.652

1.613

1.690

95.5

4.0

100.0

2

1.792

1.783

1.755

1.777

1.738

102.9

3

1.782

1.766

1.721

1.756

1.718

101.7

Positive Control

1

0.104

0.100

0.101

0.101

0.063

0.060

3.7

5.3

3.5

2

0.094

0.096

0.096

0.095

0.057

3.4

3

0.097

0.098

0.097

0.097

0.059

3.5

Test Item

1

1.698

1.650

1.668

1.672

1.634

1.687

96.7

3.4

99.8

2

1.776

1.705

1.673

1.718

1.679

99.4

3

1.811

1.749

1.798

1.786

1.748

103.4

 

 

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:
In this study and under the experimental conditions reported, the test item is non-irritant to skin.
Executive summary:

In the current study the skin irritation potential of the test item was assessed by means of the Human Skin Model Test according to OECD 439 and GLP.

In a pretest the colourless test item did not reduce MTT (test for direct MTT reduction) or did not change the colour when it was mixed with deionised water (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues were not necessary.

The main study consisted of topical exposure of the test item to the human reconstructed epidermis model followed by a cell viability test. The cell viability was 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 was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls was used to predict the skin irritation potential of the substance 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%.

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

Hereafter the skin tissues were 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.

The negative control had absorbance values well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. The positive control induced a sufficient decrease in the relative absorbance compared to the negative control, thus assuring 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 the threshold of the OECD TG, thus ensuring the validity of the study.

Treatment with the test item resulted in a mean relative absorbance value of 99.8% compared to the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritating potential.

It can be stated that in this study and under the experimental conditions reported, the test item is non-irritant to skin.