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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:
07 September - 04 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 431 without any deviation.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
dated 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: method B.40bis of the Council Regulation No. 440/2008
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
specific gravity: 1.106
Optical rotation: +1.04°
Refractive index: 1.5749

In vitro test system

Test system:
human skin model
Source species:
other: reconstituted epidermis
Cell type:
other: reconstituted epidermis (epiCS®, CellSystems®)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.60 cm2 reconstituted epidermis (epiCS®)

EXPOSURE
- The test item has been applied to the epidermal surface of 2 human skin model, during 3 minutes and during 1 hour.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 3 minutes and 1 hour after the test item application, the human epidermis was washed20 times with 20 mL of DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability is quantified by measurement of the cellular mitochondrial dehydrogenases activity. These enzymes are responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; EINECS number 206-069-5, CAS number 298-93-1)] reduction into blue formazan in the viable cells. The skin sample is placed in MTT solution of appropriate concentration (e.g. 0.3 or 1 mg/mL) for 2 hours and 55 minutes at 37°C ± 1°C. The precipitated blue formazan product is then extracted using a solvent (e.g. isopropanol), and the concentration of formazan is measured by determining the Optical Density (OD) at a wavelength between 540 and 600 nm (preferably 570 nm). The measured absorbances are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader supplied by BioTek and the validated software Gens ELISA V1.05.11 supplied by BioTek.

NUMBER OF REPLICATE TISSUES:
Duplicate skin tissues for test item, negative and positive controls

VIABILITY
Viability = (OD test item / OD negative control) x 100
For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
3 minutes and 1 hour
Number of replicates:
Duplicate skin tissues for test item, negative and positive controls

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test (3 minutes)
Value:
95.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test (1 hour)
Value:
17.01
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion
Other effects / acceptance of results:
VIABILITY
- 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 95.10% and 17.01%, respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes; 3 minutes and 1 hour after the negative control application, the viability of the human skin model has been 100%.
- Acceptance criteria met for positive control: Yes; 1 hour after the positive control application, the viability of the human skin model has been 0.62%.

Deviation to the study plan

Acceptability criteria:
The mean OD of negative control tissues for the treatment of 3 minutes and for the treatment of 1 hour were respectively 1.246 and 1.282 instead of ≥ 0.3 and ≤ 0.9 as initially scheduled.

Considering the results obtained and the fact that this values remain in the range of our historical negative control data (see appendices 2 & 3), this deviation is considered as without impact on the conclusion of the study.

Any other information on results incl. tables

Table 7.3.1/1: Skin corrosion assay: Results

 

Skin

OD

Mean OD / disc (#)

Mean OD / product

Viability %

Mean viability %

Viability difference between replicates %

Treatment: 3 min

Negative control

1

1.288

1.245

1.246

99.96

100

0.1

1.25

1.197

2

1.274

1.246

100.04

1.236

1.227

Positive control

3

0.433

0.428

0.72

34.36

57.77

46.8

0.423

0.427

4

1.069

1.011

81.17

0.967

0.997

Test item

7

1.25

1.225

1.185

98.35

95.1

6.5

1.212

1.213

8

1.165

1.144

91.85

1.147

1.12

Treatment: 1 hour

Negative control

11

1.443

1.231

1.282

96.06

100

7.9

1.113

1.137

12

1.341

1.332

103.94

1.314

1.34

Positive control

13

0.002

0.003

0.008

0.23

0.62

0.8

0.003

0.004

14

0.014

0.013

1.01

0.013

0.013

Test item

17

0.231

0.225

0.218

17.56

17.01

1.1

0.22

0.224

18

0.221

0.211

16.47

0.207

0.204

#: mean of 3 values

OD: optical density

Note:

30 minutes exposure: If the viability obtained for the test substance is greater than 50%, then it is non-corrosive.

1 hour exposure: If the viability obtained for the test substance is greater than15%, then it is non-corrosive.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required.
Executive summary:

An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item was applied as supplied, at the dose of 50 μL, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour, followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 95.10% and 17.01% versus 57.77% and 0.62%, respectively, with the positive control item (potassium hydroxide 8N).

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required.