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:
key study
Study period:
14th June 2018 - 17th July 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: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Batch: 0400

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EpiDerm Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek
- Tissue lot number: 28623

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were gently rinsed about 20 times with PBS (phosphate buffered saline). Excess PBS was removed by gently shaking the insert and blotting the bottom with blotting paper.
- Observable damage in the tissue due to washing: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of pre-warmed MTT solution was used
- Incubation time: 3 hours
- Spectrophotometer: Plate spectrophotometer
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA
- 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
Amount/concentration applied:
TEST MATERIAL
- Concentration (if solution): 25 mg + 25 μL H2O

NEGATIVE CONTROL
- Concentration (if solution): 50 μL distilled water

POSITIVE CONTROL
- Concentration (if solution): 50 μL 8 N KOH

Duration of treatment / exposure:
3 min and 60 min exposure time
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes treatment
Value:
112.9
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:
60 minutes treatment
Value:
84.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: The test material showed no non-specific MTT- reducing.
- Colour interference with MTT: The test item showed water-colouring potential in Aqua dest. and in isopropanol in the range of 570 ± 30 nm. Therefore, additional viable tissue controls were treated with the test item to determine the non-specific colour (NSCliving) and the results were corrected to the true MTT metabolic conversion (TODTT).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - mean absolute OD570 nm for 3 min experiment: 1.651; mean absolute OD570 nm for 60 min experiment: 1.896
- Acceptance criteria met for positive control: Yes - mean relative tissue viability for 60 min experiment: 6.6%
- Acceptance criteria met for variability between replicate measurements: Yes - CV: 4.4 - 12.8%

Any other information on results incl. tables

Table 1: Results of 3 min experiment

Name

Negative control

Test item

Positive control

Tissue

1

2

1

2

1

2

 

Absolute OD570

1.585

1.687

1.926

1.819

0.192

0.161

1.596

1.716

1.920

1.782

0.195

0.161

1.617

1.705

1.896

1.805

0.195

0.168

 

OD570 -

Blank Corrected

1.540

1.642

1.882

1.774

0.148

0.116

1.551

1.671

1.875

1.737

0.150

0.116

1.572

1.660

1.851

1.760

0.150

0.123

Mean OD570 of 3 Aliquots

(Blank Corrected)

1.554

1.658

1.869

1.757

0.149

0.118

SD OD570 of 3 Aliquots

0.016

0.015

0.016

0.019

0.002

0.004

Total Mean OD570 of 2

Replicate Tissues (Blank

Corrected)

 

1.606*

 

1.813

 

0.134

TODTT

 

1.813

 

SD OD570 of 2 Replicate

Tissues

0.073

0.080

0.022

Mean Relative Tissue

Viability [%]

100.0

112.9

8.3

Mean Relative Tissue

Viability [%]

- NSCliving Corrected

-

112.9

-

Coefficient Of Variation

[%]***

4.6

4.4

16.3

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is

≤ 30%.

Table 2: Results of 60 min experiment

Name

Negative control

Test item

Positive control

Tissue

1

2

1

2

1

2

 

Absolute OD570

2.007

1.778

1.747

1.476

0.187

0.148

1.996

1.803

1.760

1.475

0.187

0.151

1.987

1.803

1.763

1.468

0.186

0.148

 

OD570-

Blank Corrected

1.962

1.733

1.702

1.431

0.142

0.104

1.951

1.758

1.715

1.430

0.142

0.106

1.942

1.759

1.719

1.423

0.141

0.104

Mean OD570of 3 Aliquots

(Blank Corrected)

1.952

1.750

1.712

1.428

0.142

0.105

SD OD570of 3 Aliquots

0.010

0.015

0.009

0.004

0.001

0.002

Total Mean OD570of 2

Replicate Tissues (Blank

Corrected)

 

1.851

 

1.570

 

0.123

TODTT

-

1.570

-

SD OD570of 2 Replicate

Tissues

0.143

0.201

0.026

Mean Relative Tissue

Viability [%]

100.0

84.8

6.6**

Mean Relative Tissue

Viability [%]

- NSClivingCorrected

-

84.6

-

Coefficient Of Variation

[%]***

7.7

12.8

21.2

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the 60 min positive control < 15%,

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is

≤ 30%

Table 3: Result of the NSCliving control at 3 min

NSCliving

TVT

Negative control

Tissue

1

2

1

2

 

absolute OD570 - values

0.047

0.047

1.585

1.687

0.049

0.049

1.596

1.716

0.048

0.048

1.617

1.705

absolute OD570-

Blank corrected values

0.002

0.002

1.540

1.642

0.005

0.004

1.551

1.671

0.003

0.003

1.572

1.660

mean OD570

(mean of 3 aliquots)

0.003

0.003

1.554

1.658

total mean OD570

(mean of replicate

tissues)

 

0.003

 

1.606

SD OD570

(of the 2 replicate

tissues)

0.000

0.073

NSCliving [%]

0.2

-

Relative Tissue Viability

[%]

-

96.8

103.2

Mean Relative Tissue

Viability [%]

-

100.0

SD Tissue Viability [%]

-

4.6

CV [% Viabilities]

-

4.6

Table 4: Result of the NSCliving control at 60 min

NSCliving

TVT

Negative control

Tissue

1

2

1

2

 

absolute OD570 - values

0.048

0.048

2.007

1.778

0.053

0.050

1.996

1.803

0.048

0.048

1.987

1.803

absolute OD570-

Blank corrected values

0.003

0.003

1.962

1.733

0.008

0.005

1.951

1.758

0.003

0.003

1.942

1.759

mean OD570

(mean of 3 aliquots)

0.005

0.003

1.9552

1.750

total mean OD570

(mean of replicate

tissues)

0.004

1.851

SD OD570

(of the 2 replicate

tissues)

0.001

0.143

NSCliving[%]

0.2

-

Relative Tissue Viability

[%]

-

105.4

94.6

Mean Relative Tissue

Viability [%]

-

100.0

SD Tissue Viability [%]

-

7.7

CV [% Viabilities]

-

7.7

The test item showed no non-specific MTT-reducing but water-colouring potential in Aqua dest. And in isopropanol in the range of 570 ± 30 nm . Therefore, additional viable tissue controls were treated with the test item to determine the non-specific colour (NSCliving) and the results were corrected to the true MTT metabolic conversion (TODTT).

  

As NSClivingwas ≤ 5% relative to the negative control of living epidermis, no correction of the results was necessary.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was 50% (112.9%) after 3 min treatment and 15% (84.8%) after 60 min treatment.

The controls confirmed the validity of the study. The mean OD570nmof the two negative control tissues was 0.8 and 2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (1.896%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was 30% (4.4% - 12.8%).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed no corrosive effects and is therefore classified as 'non-corrosive'.