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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August 2017 to 22 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: suspension

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Reconstructed Human Epidermis
Cell source:
other: MatTek kit
Justification for test system used:
Description EpiDermTM (EPI-200) Reconstructed Human Epidermis
Lot Number 25842
Date of Receipt 19th Sep 2017
Morphology EPI-200 LOT#: 25842
Histological examination demonstrates human epidermis-like structure: including multiple layers (at least 4) of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) which are present under multilayered stratum corneum: 9 layers--> - PASS
Tissue thickness: 95.5 µm --> Acceptance range: >70 µm and <130 µm) - PASS
Tissue Viability: Optical Density (O.D.) values: 1.39 ± 0.046 (mean and SD of MTT value of 3 tissues exposed to H2O) -->Acceptance range: [1.0 - 3.0]- PASS
Skin Barrier Function: ET 50% of tissues exposed to 100 µL Triton X-100 1% (n=3): 7.12 h -->Acceptance range: [4.77h- 8.72h] - PASS
Sterility: No evidence of contamination during long term antibiotic and antimycotic free culture: No contamination reported --> - PASS
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek
- Tissue batch number(s): 25842
- Delivery date: 19 September 2017
- Date of initiation of testing: 25 September

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 31 minutes at room temperature and 34 minutes at 37°C, 5% CO2, ≥95% RH
- Temperature of post-treatment incubation: 42 ± 4h post-treatment incubation

REMOVAL OF TEST MATERIAL AND CONTROLS: rinsing (no details)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: no data
- Incubation time:no data
- Spectrophotometer: BMG LabTech FluoStar Optima (calibrated on 24 July 2017)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: not indicated

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: based on blanks obtained during 66 historical runs (no data provided)

NUMBER OF REPLICATE TISSUES: 3/treatment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
•A test item is considered to be an irritant (I) to skin in accordance with UN GHS Category 2 or EU R38 if the skin model viability after exposure and post-treatment incubation is ≤50%.
•A test item may be considered as a non-irritant (NI) if the skin model viability after exposure and post-treatment incubation is >50%.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mgTEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg neat


NEGATIVE CONTROL:Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) neat

POSITIVE CONTROL: 5% Sodium dodecyl sulphate (SDS)
- Concentration (if solution): 5% in water
Duration of treatment / exposure:
31 minutes at room temperature and 34 minutes at 37°C, 5% CO2, ≥95% RH
Duration of post-treatment incubation (if applicable):
42 ± 4h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
97.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Prior to the study, the required compatibility checks confirmed that the test item did not interfere with MTT and no water colouration was observed.

Acceptance criterion 1 The mean OD570 of the negative control (treated with DPBS) tissues must be ≥ 0.8 and ≤ 2.8  1.514 PASS
Acceptance criterion 2 The mean of the positive control relative percentage viability must be ≤ 20% of the mean of the negative controls.  5.097 PASS
Acceptance criterion 3 The standard deviation of viability percentages for triplicate skin models in each experimental condition must be < 18%  NC: 5.243; PC: 1.176; TA2: 12.886 PASS
Acceptance criterion 4 The mean OD of the 6 wells containing extraction solvent alone (blanks) must be ≤ 0.1.  0.162 FAIL

blank controls:
Optical Density (OD) values obtained with blanks were higher than 0.1 (0.162) causing a deviation from the acceptance criteria. However, the spectrophotometer was fully validated and had passed all required tests. The OD values for blanks observed in this study are consistent with historical data using this spectrophotometer in the XCellR8 laboratory and meet our current internal acceptance criteria of blank OD values <0.194 (mean of XCellR8 historical data, based on blanks obtained during 66 historical runs), therefore this is not considered to be an issue in the interpretation of this study data.

Any other information on results incl. tables

Viability measurements after 65 min of application and 42h (± 4h) post-incubation of test and reference items and controls. 

Condition

Tissue #

Raw data

Blank corrected data

Mean OD

% of Viability

Aliquot 1

Aliquot 2

Aliquot 1

Aliquot 2

NC

Tissue 1

1.781

1.753

1.619

1.591

1.605

106.022

Tissue 2

1.657

1.587

1.495

1.425

1.460

96.444

Tissue 3

1.663

1.614

1.501

1.452

1.477

97.534

PC

Tissue 1

0.268

0.251

0.106

0.089

0.098

6.451

Tissue 2

0.224

0.231

0.062

0.069

0.066

4.337

Tissue 3

0.226

0.234

0.064

0.072

0.068

4.502

TA2

Tissue 1

1.481

1.453

1.319

1.291

1.305

86.207

Tissue 2

1.657

1.552

1.495

1.390

1.443

95.288

Tissue 3

1.859

1.845

1.697

1.683

1.690

111.636

NC: negative control (DPBS), PC: Positive control (SDS 5%), TA2: test substance

Code

Mean of OD

SD of OD

Mean of viability (%)

SD of viability (%)

CV %

Classification

NC

1.514

0.079

100.000

5.243

5.243

Non-Irritant

PC

0.077

0.018

5.097

1.176

23.064

Irritant

TA2

1.479

0.195

97.710

12.886

13.188

Non-Irritant

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the outcome of the study the substance is considered not irritating to the skin.
Executive summary:

The skin irritation potential of the substance was assessed in vitro according to OECD Test Guideline 439.After a 65 minute exposure on the surface of the EpiDermTM reconstructed human epidermis and a 42 ± 4h post-exposure incubation time, viability of the tissues was assessed and compared to the negative control. The percentage of viability obtained was 97.710 % and therefore the substance is considered as non-irritant to the skin