Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 August 2016 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
see below:
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
see below:
Principles of method if other than guideline:
The quality criteria required for acceptance of results in the test were not fully satisfied. This is reported as a deviation. The quality criterion that was not satisfied was the standard deviation value of the % viability from the three test item treated tissues (19.3%) which marginally exceeded the upper limit of the assay acceptance criteria (≤18%). Furthermore, the results from the three test item treated tissues were unequivocally negative (individual % viability was ≥100%). Therefore, this deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
CAS RN: 15337-18-5
Purity: 96.6%
Physical state/Appearance: Yellow viscous liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed human epidermis tissues
Cell source:
other: not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and were incubated at 37 ºC, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.

MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, incubated for 3 hours at 37 °C, 5% CO2 in air, and then placed onto absorbent paper to dry. A total biopsy of the epidermis was made, the epidermis was carefully separated from the collagen matrix, and both parts (epidermis and collagen matrix) placed into labeled micro tubes containing acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation, mixed thoroughly on a vortex mixer, and then were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate samples were transferred to the appropriate wells of a pre labeled 96 well plate. Acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.
Control samples:
other: Negative Control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ and Positive Control: Sodium Dodecyl Sulphate (SDS)
Amount/concentration applied:
10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis surface.
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
Duration of post-treatment incubation (if applicable):
At the end of the exposure period each tissue was rinsed before incubating for 42 hours.
Number of replicates:
Three.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 Hours post exposure incubation period.
Value:
120.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple, which indicated that the test item did not directly reduce MTT. 

 

Assessment of Color Interference with the MTT Endpoint

The solution containing the test item was colorless. It was, therefore, unnecessary to run color correction tissues.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 120.7% after a 15 Minute exposure period and 42 Hour post exposure incubation period. It was considered unnecessary to perform the inflammatory mediator IL-1aanalysis as the results of the MTT test were unequivocal. The maintenance medium that was retained for possible analysis was discarded without evaluation.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 7.2% relative to the negative control treated tissues and the standard deviation value of the viability was 2.3%. The positive control acceptance criteria were therefore satisfied. The mean OD562 for the negative control treated tissues was 0.935 and the standard deviation value of the viability was 2.4%. The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 19.3%. The test item acceptance criterion was therefore not satisfied, but this deviation was not considered to have affected the integrity or validity of the study, as the results from the three test item treated tissues were unequivocally negative. 

 

Individual and Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.916

0.935

0.023

98

100*

2.4

0.928

99.3

0.96

102.7

Positive Control Item

0.082

0.067

0.021

8.8

7.2

2.3

0.076

8.1

0.043

4.6

Test Item

0.973

1.129

0.181

104.1

120.7

19.3

1.327

141.9

1.087

116.3

The mean viability of the negative control tissues is set at 100%

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-irritant (EU CLP Not classified for Irritation).
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. 

 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. The optical density was measured at 562 nm. The relative mean viability of the test item treated tissues was 120.7% after the 15 minute exposure period and 42 hours post exposure incubation period.

 

The relative mean tissue viability for the positive control treated tissues was 7.2% relative to the negative control treated tissues and the standard deviation value of the viability was 2.3%.  The positive control acceptance criteria were therefore satisfied.  The mean OD562 for the negative control treated tissues was 0.935 and the standard deviation value of the viability was 2.4%.  The negative control acceptance criteria were therefore satisfied.

 

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 19.3%.  The test item acceptance criterion was therefore not satisfied and this is reported as a study plan deviation.

 

The test item was classified as non-irritant (EU CLP Not classified for Irritation).