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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:
27th April 2018 to 23rd May 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: Human Skin Model Test)
Version / remarks:
29 July 2016
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
May 30, 2008
Deviations:
not specified
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol for : In Vitro EpiDermTM Skin Corrosion Test (EIP-200-SCT)
Version / remarks:
07/11/2014
Deviations:
not specified
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PharmaZell, batch no. 12/18
- Expiration date of the lot/batch: not provided
- Purity test date: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble. Assumed stable within the short time-frame of the test
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: unknown

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: notne
- Final preparation of a solid: none

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek
Details on test system:
SKIN DISC PREPARATION
- Procedure used: skin supplied in ready-to-use kits. The tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 ± 1°C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C for both the 3 minute and 60 minute tests

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 20 times for both 3 minute and 60 minute tests
- Observable damage in the tissue due to washing: non specified
- Modifications to validated SOP: none stated

DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:] : MTT
- Spectrophotometer: plate
- Wavelength: 570 nm
- Filter: not stated
- Filter bandwidth: not stated

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 mg + 25 µL H2O

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N KOH
Duration of treatment / exposure:
3 mins and 60 minutes
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
2

Test animals

Species:
other: Not applicable

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of replicates 1+2 in the 3 minute test
Value:
98.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of replicates 1+2 in the 60 minute test
Value:
96.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Non-corrosive
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: test item did not cause direct MTT reduction
- Colour interference with MTT: none


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline:
OD570 for negative control (3 minute test) - mean 1.895, with SD 0.313 (10 samples)
OD570 for negative control (60 minute test) - mean 1.867, with SD 0.261 (11 samples)
Historical control data were generated from 2015-2016

Any other information on results incl. tables

Pre-experiments

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixture of 25 mg test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equaled 0%.

Experiment

3 minute experiment

Name

Negative Control

Test item

Positive control

Tissue 

1

2

1

2

1

2

Absolute OD570

1.736

1.769

1.697

1.722

0.080

0.083

1.713

1.715

1.726

1.735

0.079

0.081

1.770

1.762

1.724

1.725

0.084

0.088

OD570(Blank Corrected)

1.691

1.724

1.652

1.678

0.035

0.039

1.669

1.670

1.682

1.691

0.035

0.036

1.725

1.718

1.680

1.680

0.040

0.044

Mean OD570of 3 aliquots (Blank corrected)

1.695 

1.704 

1.671  

1.683 

0.037 

0.040

SD OD570of 3 aliquots

0.028 

0.030 

0.016 

0.007 

0.003 

0.004

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.700*

1.677 

0.038

SD OD570 of 2 Replicate Tissues

0.006 

0.008 

0.002

Mean Relative Tissue Viability [%]

100.0 

98.7 

2.2

Coefficient Of Variation [%]***

0.4 

0.5 

5.1

*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%

60 minute experiment

Name

Negative Control

Test item

Positive control

Tissue 

1

2

1

2

1

2

Absolute OD570

1.786

1.922

1.867

1.713

0.121

0.145

1.780

1.887

1.854

1.716

0.120

0.139

1.770

1.923

1.827

1.693

0.126

0.146

OD570(Blank Corrected)

1.741

1.877

1.822

1.668

0.076

0.100

1.734

1.842

1.809

1.671

0.075

0.093

1.725

1.878

1.782

1.648

0.080

0.100

Mean OD570of 3 aliquots (Blank corrected)

1.733

1.865

1.804

1.662

0.077

0.098

SD OD570of 3 aliquots

0.008

0.021

0.021

0.012

0.003

0.004

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.799*

1.733

0.087

SD OD570 of 2 Replicate Tissues

0.093

0.101

0.015

Mean Relative Tissue Viability [%]

100.0

96.3

4.9**

Coefficient Of Variation [%]***

5.2

5.8

16.9

*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:
GHS criteria not met
Remarks:
non-corrosive
Conclusions:
The test item is classified as “non-corrosive“.