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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:
13.12.2016-01.03.2017
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: Human Skin Model Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
- Pre-incubation: the inserts containing the tissues were set into the incubator at 37 ± 1°C, 5.0% CO2 / 95% air for 1 hour.
- For each experiment (“3 minutes” and “1 hour”), the plates were stored in the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2.
- MTT medium: the tissues were incubated with MTT medium for 3 h at 37 ± 1°C, 5.0% CO2 / 95% air.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg test item + 25 µL H2O
Duration of treatment / exposure:
3 and 60 minutes treatment
Number of replicates:
2 replicates for each treatment period (3 min and 60 min exposure time)

Test system

Details on study design:
Details of the test procedure
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (MatTek, EPI-200-SCT)
- Conditions of exposure: 37 ± 1 °C, 5% CO2
- Washing: the tissue was gently rinsed about 20 times with PBS
- Number of tissue replicates used per test chemical and controls: 2
- MTT assay: incubation with 0.3 mL of MTT solution for 3 hours at 37 ± 1 °C, 5% CO2)

- Data evaluation:
Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%) according to the following Prediction Model
In step 1:
< 50% after 3 min exposure: predicted as corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure: predicted as corrosive (a combin
ation of optional sub-categories 1B and 1C)
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure: predicted as Non-Corrosive
In step 2:
< 25% after 3 min exposure: optional Sub-category 1A
≥ 25% after 3 min exposure: a combination of optional Sub-categories 1B and 1C

- Historical data negative control: Mean Absorption: 1.895 (3 min), 1.867 (1 h); Standard Deviation: 0.313 and 0.261, respectively
- Historical data positive control: Mean Viability: 6.1% (1 h); Standard Deviation: 1.99%

- The test meets acceptance criteria if:
- 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,
- mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15%,
- coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is <= 30%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min. incubation time
Value:
>= 87.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not corrosive to skin
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour incubation time
Value:
>= 17.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: borderline result
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour incubation time
Value:
>= 20.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: repeated due to a borderline result
Remarks:
no corrosive effects
Other effects / acceptance of results:
The controls confirmed the validity of the study. The mean OD570 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% (5.4% and 10.7%) 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% (2.8% - 13.3%).

Any other information on results incl. tables

Results of the 3 min experiment:

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

Absolute OD570

2.006

1.759

1.723

1.516

0.098

0.180

2.021

1.738

1.839

1.490

0.097

0.182

2.055

1.751

1.883

1.525

0.099

0.189

OD570- Blank Corrected

1.963

1.715

1.680

1.473

0.055

0.137

1.977

1.695

1.796

1.447

0.054

0.139

2.011

1.708

1.840

1.482

0.056

0.146

Mean OD570of 3 Aliquots (blank corrected)

1.984

1.706

1.772

1.467

0.055

0.141

SD OD570 of 3 Aliquots

0.025

0.026

0.078

0.029

0.024

0.024

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.845

1.619

0.098

TODTT

-

1.608

-

SD OD570 of 2 Replicate Tissues

0.197

0.215

0.061

Mean Relative Tissue
Viability [%]

100.0

87.8

5.3

NSMTT-corrected mean relative tissue viability [%]

-

87.2

-

Coefficient Of Variation [%]

10.7

13.3

62.2

Results of the 60 min experiment:

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

Absolute OD570

1.627

1.683

0.346

0.275

0.155

0.110

1.601

1.733

0.339

0.278

0.155

0.111

1.641

1.721

0.339

0.270

0.156

0.113

OD570- Blank Corrected

1.582

1.638

0.302

0.230

0.110

0.065

1.556

1.688

0.294

0.233

0.110

0.066

1.596

1.676

0.294

0.226

0.111

0.068

Mean OD570of 3 Aliquots (blank corrected)

1.578

1.667

0.296

0.230

0.111

0.066

SD OD570 of 3 Aliquots

0.020

0.034

0.025

0.025

0.025

0.025

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.623

0.263

0.088

TODTT

-

0.289

-

SD OD570 of 2 Replicate Tissues

0.063

0.047

0.031

Mean Relative Tissue
Viability [%]

100.0

16.2

5.4

NSMTT-corrected mean relative tissue viability [%]

-

17.4

-

Coefficient Of Variation [%]

3.9

17.9

35.5

Because of the borderline result, a second experiment was conducted.

Results of the second 60 min experiment:

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

Absolute OD570

1.644

1.707

0.329

0.393

0.209

0.229

1.700

1.672

0.352

0.400

0.213

0.228

1.826

1.598

0.351

0.383

0.213

0.232

OD570- Blank Corrected

1.601

1.664

0.285

0.349

0.166

0.185

1.657

1.628

0.309

0.357

0.169

0.184

1.783

1.554

0.308

0.340

0.169

0.189

Mean OD570of 3 Aliquots (blank corrected)

1.680

1.616

0.301

0.349

0.168

0.186

SD OD570 of 3 Aliquots

0.093

0.055

0.027

0.025

0.024

0.024

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.648

0.325

0.177

TODTT

-

0.351

-

SD OD570 of 2 Replicate Tissues

0.046

0.034

0.013

Mean Relative Tissue
Viability [%]

100.0

19.7

10.7

NSMTT-corrected mean relative tissue viability [%]

-

20.9

-

Coefficient Of Variation [%]

2.8

10.4

7.2

Applicant's summary and conclusion

Conclusions:
The test item showed no corrosive effects. The test item is classified as “non-corrosive“.
Executive summary:

In an in vitro study, the skin corrosion potential of the test item was assessed with the Reconstructed Human Epidermis (RhE)

test method according to OECD 431 and EU-Method B.40 and in compliance to GLP.

The test item was applied topically and cytotoxic effects to the stratum corneum after a short time exposure were determined. 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 (NSMTT) potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT. After 3 min treatment, NSMTT was 0.6%, after 60 min treatment, NSMTT was -1.2%. The results were corrected accordingly. Moreover, the test item showed no colouring potential after mixture with aqua dest. but colouring was detected after mixture with isopropanol. Since there was no relevant absorption in the range of 570 ± 30 nm, NSCliving(non-specific colour of additional viable tissues) was not determined.

The corrosivity potential was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%). According to the Prediction Model: a mean relative tissue viability of ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure conclude to a non-corrosive effect of the test item. Due to a borderline result (17.4%) the 60 min treatment period was repeated. The mean relative tissues viability (% of negative control) was 87.2 % (NSMTT-redcuced) after 3 min treatment and was 17.4 % and 20.9% after 60 min treatment.

According to the Prediction Model, the test item showed no corrosive effects.