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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:
16 Nov 2017 - 01 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted in 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200)
Justification for test system used:
The EpiDermTM 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: EpiDerm Skin Model (EPI-200, MatTek Corporation, Ashland MA, U.S.A.)
- Tissue batch numbers: 25802 and 25857

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after exposure the tissues were removed from the 6-well plate with forceps; the tissues were gently rinsed about 20 times with PBS (phosphate buffered saline) using a wash bottle, to remove any residual test item; excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: plate spectrophotometer (not further specified)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) for the two tissue batches were 1.82 ± 0.198 and 1.751 ± 0.073, respectively (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 1% Triton X-100. The ET-50 value for the two tissue batches were 5.7 and 7.19 h, respectively (acceptance criteria: 4.77 - 8.72 h).
- Contamination: The cells used to produce the EpiDermTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 2 x 2 replicates (3 and 60 min) for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh and killed tissues
- No. of replicates: 2 each for fresh and killed tissues
- Method of calculation used: NSCliving [%] = [ODTVT/ODNK]*100 and NSCkilled [%] = [ODTKT/ODNK]*100

NSCliving = non-specific color of additional viable tissues
ODTVT = optical densitiy of additional test item treated killed tissue without MTT staining
ODNK = optical densitiy of negative control of living tissues
NSCkilled = non-specific color of additional additional killed tissues

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

PREDICTION MODEL / DECISION CRITERIA
- The test item 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
- Amount applied: 25 mg ; applied directly atop the EpiDermTM tissue using an application spoon avoiding compression of the test item; to ensure good contact with the skin the test item was moistened with 25 µL H2O; the test item was spread to match the size of the tissue

VEHICLE
- not applicable

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N
Duration of treatment / exposure:
3 min and 60 min
Duration of post-treatment incubation (if applicable):
3 h
Number of replicates:
duplicates for each treatment and control group

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
3 min exposure
Value:
86.2
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
Remarks:
mean value of 2 tissues
Run / experiment:
60 min exposure
Value:
99.2
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:
- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: The test item showed non-specific MTT-reducing potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT (NSMTT) and the results were corrected to the true MTT metabolic conversion (TODTT).
- Colour interference with MTT: the test item showed water-colouring potential in aqua dest. and 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). As NSCliving was ≤ 5% relative to the negative control of living epidermis, no correction of the results was necessary.
Since correction of the results with the NSCliving control was not necessary, double correction with the NSCkilled control was not required.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Laboratory technical proficiency with the test system according to OECD 431 was demonstrated at Eurofins Biopharma Product Testing Munich GmbH as document in the Eurofins Biopharma Product Testing Munich GmbH “Zusammenfassung Ergebnisse Subkategorisierung in vitro Hautkorrosion mit EpiDerm”.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD570 nm of the two negative control tissues of the 3 min and 60 min treatment period is between 0.8 and 2.8
- Acceptance criteria met for positive control: the mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15%,
- Acceptance criteria met for variability between replicate measurements: the coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%

Any other information on results incl. tables

Table 2. Results of 3 min experiment

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

 

Absolute OD570

1.779

1.692

1.767

1.218

0.137

0.151

1.873

1.792

1.850

1.331

0.141

0.155

1.890

1.781

1.859

1.334

0.145

0.157

OD570 -

Blank Corrected

1.732

1.646

1.720

1.172

0.091

0.105

1.827

1.746

1.803

1.284

0.095

0.109

1.844

1.734

1.813

1.287

0.098

0.111

Mean OD570 of 3 Aliquots (Blank Corrected)

 

1.801

 

1.709

 

1.779

 

1.248

 

0.095

 

0.108

SD OD570 of 3 Aliquots

0.060

0.055

0.051

0.066

0.004

0.003

Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)

 

1.755*

 

1.513

 

0.101

TODTT NSMTT

-

1.507

-

TODTT NSMTT und

NSCliving

-

1.507

-

SD OD570 of 2 Replicate Tissues

0.065

0.376

0.010

Mean Relative Tissue Viability [%]

100.0

86.2

5.8

Mean Relative Tissue Viability [%]

- NSMTT Corrected

 

-

 

85.9

 

-

Mean Relative Tissue Viability [%] - NSMTT and NSCliving Corrected

 

-

 

85.9

 

-

True Tissue Viability [%]

-

double correction not necessary

-

Coefficient Of Variation [%]***

3.7

24.8

9.5

Table 3:  Results of 60 min experiment

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

 

Absolute OD570

1.710

1.771

1.779

1.715

0.072

0.118

1.767

1.907

1.859

1.777

0.080

0.126

1.781

1.907

1.864

1.767

0.075

0.122

OD570 -

Blank Corrected

1.664

1.725

1.733

1.669

0.026

0.072

1.721

1.861

1.813

1.731

0.033

0.079

1.735

1.861

1.817

1.721

0.029

0.075

Mean OD570 of 3 Aliquots (Blank Corrected)

1.707

1.815

1.788

1.707

0.029

0.075

SD OD570 of 3 Aliquots

0.038

0.078

0.047

0.033

0.004

0.004

Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)

 

1.761*

 

1.747

 

0.052

TODTT NSMTT

-

1.737

-

TODTT NSMTT und NSCliving

-

1.737

-

SD OD570 of 2 Replicate Tissues

0.077

0.057

0.033

Mean Relative Tissue Viability [%]

100.0

99.2

3.0**

Mean Relative Tissue Viability [%]

- NSMTT Corrected

 

-

 

98.6

 

-

Mean Relative Tissue Viability [%] - NSMTT and NSCliving Corrected

 

-

 

98.6

 

-

True Tissue Viability [%]

-

double correction not necessary

-

Coefficient Of Variation [%]***

4.4

3.3

62.5

*         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%

Applicant's summary and conclusion

Interpretation of results:
other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: not classified
Under the conditions of the Reconstructed Human Epidermis test the test substance does not possess any skin corrosion potential.