Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 February to 19 March, 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: human derived epidermis keratinocytes
Cell source:
other: reconstructed human epidermis (RhE)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Negative control (live tissue) D-PBS 20 μL
Positive control (live tissue) 5% (w/v) SDS 20 μL
Test item (live tissue) 20 ± 2 mg
Duration of treatment / exposure:
exposure time: 15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
recovery period: 42 ± 1 hour
Number of replicates:
Negative control (Live tissue) D-PBS: 3 replicates
Positive control (Live tissue) 5% (w/v) SDS: 3 replicates
Test item (Live tissue): 3 replicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: mean cell viability %
Value:
95
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
PRELIMINARY TEST
No colour change was noted in the MTT solution at the end of the incubation period, indicating that the test item could not direct interact with MTT.
A clear solution was observed, indicating that the test item has not a potential interfering ability.
Based on the results obtained, no additional control was added in the Main Assay.

MAIN ASSAY
The mean Optical Density of the blank controls was found to be 0.036, within the maximum acceptable value (0.1). The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability (Standard Deviation of the % viability lower or equal to 18), in agreement with guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100 % of cell viability.
Positive control results indicated an appropriate cell death with an acceptable relative cell viability (2% of the negative control value). Variability between replicates gave also the expected value (SD of%viability = 1.0). Based on the stated criteria, mean viability, expressed as percentage of the negative control, lower or equal to 40% and standard deviation of % viability equal or lower than 18, the study was accepted as valid.
The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 95% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 8.5 (lower than 18, as stated in the study protocol).

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP Regulation (EC) No. 1272/2008
Conclusions:
The test item does not induce cell death in reconstructed human epidermis (RhE) in the EpiSkin model. The mean cell viability was found to be 95 %, therefore, the test item is not irritating/corrosive to skin.
Executive summary:

The potential of the test item to induce skin irritation was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EpiSkin™ according to the OECD Guideline 439 (2015). Ability of the test item to impair cell viability following an exposure period of 15 minutes and a subsequent 42 ± 1 hour recovery period was tested. A colorimetric measurement of MTT reduction (blue formazan salt) was used as an index for cell viability. A preliminary test was carried out to evaluate the compatibility of the test item with the test system. Firstly, the test item was assayed for the ability of reducing MTT per se. No colour change was noted in the MTT solution at the end of the incubation period, indicating that the test item could not direct interact with MTT. Secondly, the test item was assayed for its ability to colour water per se. A colourless solution was observed, indicating that the test item does not have potential interfering ability. Based on these results, no additional control was added in the Main Assay. In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 ± 2 mg/epidermis unit, each measuring 0.38cm2 (treatment level: 53 mg/cm2). A positive control (5% (w/v) sodium dodecyl sulphate solution in water) and a negative control (Dulbecco’s phosphate buffered saline (D-PBS)) were concurrently tested.

The negative control provided the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability (Standard Deviation (SD) of % viability lower or equal to 18), as indicated in the guideline, representing 100 % cell viability. The positive control caused the expected cell death (2% cell viability compared to the negative control) and variability (SD of % viability equal to 1.0). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18), the assay was regarded as valid. The test item did not induce cell death in any replicate. The mean cell viability after subtraction of the blank solution was 95% compared to the negative control. Intra-replicate variability was acceptable (SD of % viability value equal to 8.5 (lower than the maximum of 18)). Based on these results, the test item is not classified as irritating to the skin (UN GHS No Category).