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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:
weight of evidence
Study period:
Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

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:
Landesamt für Umwelt Rheinland-Pfalz

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish

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

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number: 28684

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: one washing step with PBS

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

NUMBER OF REPLICATE TISSUES: two tissues per exposure time and test group (12 tissues per test)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed at - 20 °C
- N. of replicates: two killed control tissues per exposure time were treated with the test substance and the negative control, respectively
- Method of calculation used: In case of direct MTT reduction by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the tissues treated with the test substance (mean corrected OD570 KC). Since killed tissues might still have a residual enzyme activity that is able to produce some formazan net, OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero, it is subtracted from the respective mean OD570 to result in the mean corrected OD570 KC. The mean corrected OD570 KC represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

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

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): 25 µL

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N
Duration of treatment / exposure:
3 min and 1 h
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure period
Value:
95.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 h exposure period
Value:
105.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Table 2: Summary of results after 3 minutes exposure period

Test substance identification

 

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

2.016

1.996

2.006

 

 

Viability [% of NC]

100.5

99.5

100.0

0.7

0.7

KC tissues

Mean OD570

0.114

0.092

0.103

 

 

Viability [% of NC]

5.7

4.6

5.1

0.8

15.4

Test substance

Viable tissues

Mean OD570

1.995

1.849

1.922

 

 

Viability [% of NC]

99.5

92.2

95.8

5.1

5.4

KC tissues *

Mean OD570

KC NC corrected

0.011

0.000

0.006

 

 

Viability [% of NC]

0.56

0.00

0.28

 

 

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

95.5

PC

Viable tissues

Mean OD570

0.254

0.216

0.235

 

 

Viability [% of NC]

12.7

10.8

11.7

1.3

11.4

*negative values are set to zero for further calculation

Table 3: Summary of results after 1 hour exposure period

Test substance identification

 

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Viable tissues

Mean OD570

1.854

1.875

1.864

 

 

Viability [% of NC]

99.4

100.6

100.0

0.8

0.8

KC tissues

Mean OD570

0.085

0.092

0.088

 

 

Viability [% of NC]

4.5

4.9

4.7

0.3

5.6

Test substance

Viable tissues

Mean OD570

1.941

2.030

1.986

 

 

Viability [% of NC]

104.1

108.9

106.5

3.4

3.2

KC tissues

Mean OD570

KC NC corrected

0.023

0.019

0.021

 

 

Viability [% of NC]

1.2

1.0

1.1

0.1

11.9

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

105.4

PC

Viable tissues

Mean OD570

0.097

0.106

0.101

 

 

Viability [% of NC]

5.2

5.7

5.4

0.3

6.3

Table 4: Historic control data of NC and PC of skin corrosion test

 

 

Exposure Time

Period

Mean

SD

Mean + 2 SD

Mean – 2 SD

NC

OD570

3 minutes

Jan 2017 – Jan 2019

1.728

0.216

2.160

1.295

60 minutes

1.724

0.252

2.227

1.220

PC

3 minutes

0.222

0.071

0.363

0.081

60 minutes

0.097

0.031

0.159

0.036

Relative viability [%]

3 minutes

13.0

4.2

21.3

4.6

60 minutes

5.7

1.8

9.3

2.2

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
no indication of skin corrosion
Conclusions:
Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin corrosion potential in the EpiDerm™ in vitro skin corrosion test under the test conditions chosen.
Executive summary:

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of ca. 25 μL bulk volume (about 7 mg) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

For the skin corrosion test, 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/post-incubation 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 following results were obtained in the EpiDerm™ skin corrosion test:

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 95.5%, and it was 105.4% after an exposure period of 1 hour.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin corrosion potential in the EpiDerm™ in vitro skin corrosion test under the test conditions chosen.