Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 November 2016 - 13 January 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)
Version / remarks:
January 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
solid: crystalline
Details on test material:
- Storage condition of test material: Store in cool dark place at room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Source strain:
other: Not applicable.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 17-EKIN-002
- 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Approximately 10 mg of the test item were applied and spread to the wetted triplicate tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5°C

PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT:
MTT Salt Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of thecellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
For this purpose the colourless test item (approximately 10 mg) was mixed with 90 μL of deionised water in a preexperiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
The colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the test item in water at 570 nm was not required and consequently not performed. An additional test with viable tissues (normal test procedure but without MTT addition) did not have to be performed.
Assessment of Color Interference with the MTT endpoint:
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 10 mg of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture will be incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution with 10 μL of DMEM was used as the control.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

PRE-INCUBATION:
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 23 hours.

TREATMENT:
The test item tissues were wetted with 5 μL of deionised water. The negative control, positive control and the test item were added to the inserts atop the concerning EpiSkin™ triplicate tissues. Exposure period of the tissues in 12-well plates was 15 minutes at room temperature.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

MTT ASSAY:
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for nearly 3 hours while shaking at room temperature.
Per tissue sample 2 × 200 μL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. Isopropanol was used for blank measurement.OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1)with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

DECISION CRITERIA:
For the current test, a test item is considered irritant if the mean relative tissue viability of three individual tissues is reduced to < 50% of the negative control.
For the current test, a test item is considered non-irritant if the mean relative tissue viability of three individual tissues is > 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test material:
- Amount applied: approximately 10 mg
Duration of treatment / exposure:
15-Minute exposure period and 42 hours post-exposure incubation period.
Number of replicates:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: relative mean viability
Value:
70.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The relative mean tissue viability compared to the negative control tissues (100%).
Other effects / acceptance of results:
Direct MTT Reduction and colour interference:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.
Test Item:
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 70.9% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Quality Criteria:
After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 8.1% (< 40%) thus ensuring the validity of the test system.
The rel. standard deviations between the % variability values of the test item, the positive and negative controls were below 16% (≤ 18%), thus ensuring the validity of the study.

Any other information on results incl. tables

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item

OD570 of

tissues

Mean OD570

of triplicate

tissues

Relative SD (%)

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

Negative

Control Item

1.111

1.053

8.2

105.5

100

1.094

103.9

0.954

90.6

Positive Control Item

0.074

0.086

15.9

7.0

8.1

0.082

7.8

0.101

9.6

Test Item

0.757

0.747

7.9

71.9

70.9

0.683

64.8

0.800

76.0

OD = Optical Density

SD = Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:
other: Not a skin irritant.
Remarks:
According to EU CLP Regulation (EC) No. 1272/2008 and its amendments.
Conclusions:
The substance is not a skin irritant tested in an OECDTG 439 because the mean relative tissue viability was 70%, which is > 50% the substance is considered not to be a skin irritant.
Executive summary:

The possible skin irritation potential of the substance was tested in an in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according

to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 10 mg test substance. After 42 hours post incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)

MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliablenegative and positive controls were included.The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 8.1%. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 70.9%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment, the substance is considered not to be a skin irritant.