Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Feb 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:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid, waxy, white to yellowish
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0017319511
- Expiration date of the batch: 18 Dec 2018
- Purity: > 99 %
- pH value: ca. 6
- Physical state / color: Solid, wxy / white to yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To improve application, the solid waxy test substance was heated at about 50 °C for about 15 minutes. Before application, the test substance was cooled down to room temperature.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The Skin Corrosion Test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure. The test is designed to predict a skin corrosion or irritation potential of a chemical by using the three-dimensional human epidermis model EpiDerm. After application of the test material to the stratum corneum surface of the EpiDerm tissue, the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow-colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue-colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. The optical density of the extracts of tissues treated with the test substance is compared to negative control values from tissues and expressed as relative tissue viability.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number(s): 25882

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. After incubation, the tissues were washed with PBS to stop the MTT incubation.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.9 mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: filter wavelength 570 nm without reference filter

NUMBER OF REPLICATE TISSUES: two tissues per exposure time

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
Duration of treatment / exposure:
3 min and 1 h
Number of replicates:
2 tissues per exposure time

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure period
Value:
104.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 h exposure period
Value:
59.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance

Identification

 

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.789

1.779

1.784

 

 

Viability

[% of NC]

100.3

99.7

100.0

0.4

0.4

KC tissues

Mean OD570

0.085

0.083

0.084

 

 

Viability

[% of NC]

4.8

4.7

4.7

0.1

1.7

Test substance

Viable tissues

Mean OD570

1.833

1.930

1.882

 

 

Viability

[% of NC]

102.7

108.2

105.5

3.9

3.7

KC tissues

Mean OD570

KC NC corrected

0.020

0.005

0.012

 

 

Viability

[% of NC]

1.1

0.3

0.7

0.6

83.7

Final relative mean viability of tissues after KC correction [% of NC]

104.8

 

 

PC

Viable tissues

Mean OD570

0.179

0.185

0.182

 

 

Viability

[% of NC]

10.0

10.4

10.2

0.2

2.3

Table 2: Exposure period 1 h: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance

Identification

 

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.479

1.668

1.574

 

 

Viability

[% of NC]

94.0

106.0

100.0

8.5

8.5

KC tissues

Mean OD570

0.092

0.069

0.080

 

 

Viability

[% of NC]

5.8

4.4

5.1

1.0

20.3

Test substance

Viable tissues

Mean OD570

1.160

0.856

1.008

 

 

Viability

[% of NC]

73.7

54.4

64.1

13.7

21.3

KC tissues

Mean OD570

KC NC corrected

0.062

0.089

0.075

 

 

Viability

[% of NC]

3.9

5.6

4.8

1.2

24.9

Final relative mean viability of tissues after KC correction [% of NC]

59.3

 

 

PC

Viable tissues

Mean OD570

0.065

0.089

0.077

 

 

Viability

[% of NC]

4.1

5.7

4.9

1.1

22.5

Applicant's summary and conclusion

Interpretation of results:
other: No skin corrosion potential observed.
Conclusions:
No prediction can be made for skin corrosion according to GHS criteria based on the results of this in vitro study alone.
Executive summary:

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of 50 µL undiluted test substance to a reconstructed three-dimensional, human epidermis model (EpiDerm).

The test substance could not be homogeneously distributed on the whole application area. Therefore, a metal pin covered with 50 µL undiluted, at ca. 50 °C heated test substance was applied covering the whole tissue surface. Before application, the test substance was cooled down to room temperature.

Two EpiDerm tissues were incubated with the test substance for 3 minutes and 1 hour each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced. The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 104.8 %, and it was 59.3 % after an exposure period of 1 hour.