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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:
2018-01-16 to 2018-01-18
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:
29 July 2016
Qualifier:
according to
Guideline:
other: Council Regulation 440/2008, Method B.40 BIS: “In Vitro Skin Corrosion: Human Skin Model Test”, May 30, 2008
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm; Version 07/11/2014
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 h MTT incubation
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min. treatment
Value:
97.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100.0
Positive controls validity:
valid
Remarks:
1.5
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min. treatment
Value:
106.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100.0
Positive controls validity:
valid
Remarks:
5.2
Other effects / acceptance of results:
Value Cut off pass/fail
Mean Absolute OD570 nm NK
(3 min Experiment) 1.609 0.8 ≤ NK ≤ 2.8 pass
Mean Absolute OD570 nm NK
(60 min Experiment) 1.670 0.8 ≤ NK ≤ 2.8 pass
Mean Relative Tissue Viability [%] of
PC (60 min experiment) 1.5 < 15% pass
CV [%]
(in the range of 20 – 100% viability) 7.8% - 20.1% ≤ 30% pass

Any other information on results incl. tables

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). The test item showed no water-colouring potential.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50% (110.7%, NSMTT-corrected) after 3 min treatment and ≥ 15% (97.6%, NSMTTcorrected) after 60 min treatment.

The controls confirmed the validity of the study. The mean OD570nm of 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% (1.5%) 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% (7.8% - 20.1%).

Results of 3 minExperiment

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

Absolute OD570

1.818

1.381

1.687

1.694

0.130

0.122

1.854

1.399

1.702

1.704

0.129

0.125

1.823

1.380

1.712

1.789

0.129

0.125

OD570-
Blank Corrected

1.773

1.335

1.642

1.648

0.085

0.077

1.808

1.354

1.656

1.659

0.083

0.079

1.777

1.334

1.666

1.744

0.084

0.079

Mean OD570of 3 Aliquots (Blank Corrected)

1.786

1.341

1.655

1.684

0.084

0.079

SD OD570 of 3 Aliquots

0.019

0.011

0.012

0.052

0.001

0.001

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.564*

1.669

0.081

TODTT

 -

1.731

 -

SD OD570 of 2 Replicate Tissues

0.315

0.021

0.004

Mean Relative Tissue
Viability [%]

100.0

106.8

5.2

Mean Relative Tissue Viability [%]
- NSMTT Corrected

 -

110.7

 -

Coefficient Of Variation [%]***

20.1

1.2

4.8

*     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%.

Results of the NSMTT control of 3 min Experiment

NSMTT

KU

KT

Negative Control

Tissue

1

2

1

2

1

2

absolute OD570 -values

0.576

0.495

0.449

0.501

1.818

1.381

0.590

0.490

0.460

0.497

1.854

1.399

0.597

0.493

0.445

0.497

1.823

1.380

OD570(Blank Corrected)

0.531

0.450

0.404

0.455

1.773

1.335

0.545

0.445

0.415

0.451

1.808

1.354

 

0.552

0.448

0.399

0.452

1.777

1.334

mean OD570
(mean of 2 aliquots)

0.538

0.448

0.409

0.453

1.791

1.344

total mean OD570
(mean of the replicate tissues)

0.493

0.431

1.568*

SD OD570(of the replicate tissues)

0.064

0.031

0.315

NSMTT [%]

-3.92

 -

Relative Tissue Viability [%]

 -

114.2

85.8

Mean Relative Tissue Viability [%]

 -

100.0

SD Tissue Viability [%]

 -

20.1

CV [% Viabilities]

 -

20.1

Results of 60 min Experiment

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

Absolute OD570

1.576

1.710

1.415

1.817

0.070

0.069

1.579

1.783

1.419

1.816

0.072

0.069

1.587

1.787

1.494

1.822

0.072

0.069

OD570-
Blank Corrected

1.531

1.665

1.370

1.771

0.025

0.024

1.533

1.738

1.374

1.771

0.027

0.024

1.542

1.742

1.449

1.776

0.027

0.024

Mean OD570of 3 Aliquots (Blank Corrected)

1.535

1.715

1.397

1.773

0.026

0.024

SD OD570 of 3 Aliquots

0.006

0.043

0.044

0.003

0.001

0.000

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.625*

1.585

0.025

TODTT

 -

1.586

 -

SD OD570 of 2 Replicate Tissues

0.127

0.266

0.002

Mean Relative Tissue
Viability [%]

100.0

97.5

1.5**

Mean Relative Tissue Viability [%]
- NSMTT Corrected

 -

97.6

 -

Coefficient Of Variation [%]***

7.8

16.8

6.3

*           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%.

Results of the NSMTT control of 60 min Experiment

NSMTT

KU

KT

Negative Control

Tissue

1

2

1

2

1

2

absolute OD570 -values

0.106

0.095

0.087

0.113

1.576

1.710

0.107

0.095

0.088

0.112

1.579

1.783

0.106

0.094

0.087

0.112

1.587

1.787

OD570(Blank Corrected)

0.061

0.049

0.041

0.068

1.531

1.665

0.062

0.049

0.043

0.067

1.533

1.738

 

0.061

0.049

0.042

0.066

1.542

1.742

mean OD570
(mean of the aliquots)

0.061

0.049

0.042

0.067

1.532

1.702

total mean OD570
(mean of the replicate tissues)

0.055

0.055

1.617*

SD OD570(of the replicate tissues)

0.008

0.018

0.120

NSMTT [%]

-0.04

 -

Relative Tissue Viability [%]

 -

97.7

108.6

Mean Relative Tissue Viability [%]

 -

103.1

SD Tissue Viability [%]

 -

7.6

CV [% Viabilities]

 -

7.4

 

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive
Conclusions:
In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non-corrosive“.
Executive summary:

In the present study the skin corrosivity potential of the test item was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDerm, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls.

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). The test item showed no water-colouring potential.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was 50% (110.7%, NSMTT-corrected) after 3 min treatment and  15% (97.6%, NSMTT-corrected) after 60 min treatment.