Registration Dossier

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase: 04 September 2017; End of experimental phase: 18 September 2017; Study completion 06 February 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted on 28 July 2015
GLP compliance:

Test material

Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
Cell type:
other: reconstructed human epidermis (RhE)
Details on test system:
Commercial Name: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batch: 17-EKIN-037 (alive tissues) and 17-EKIN-015 (killed tissues)
Arrived at RTC on: 12 Sep 2017 and 11 April 2017
Control samples:
yes, concurrent negative control
Amount/concentration applied:
Negative control: D-PBS 20 μL
Positive control: 5% (w/v) SDS 20 μL
Test item (Basic Orange 22): 20 ± 2 mg
Duration of treatment / exposure:
An exposure time of 15±0.5 minutes
Duration of post-treatment incubation (if applicable):
A 42 ± 1 hour recovery period
Number of replicates:
Negative control, Positive control, Test item (live tissues): 3 replicates
Negative control Test item (additional control): 2 replicates

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The mean cell viability of the test item treated tissues, after the blank subtraction, was 118%.
Based on the results obtained, the test item Basic Orange 22 is classified as non irritant to the skin.
Executive summary:

The potential of the test item Basic Orange 22 to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epi-dermis (RhE) model named EPISKINTM. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se.An orange suspension with orange precipitate was noted in the MTT solution at the end of the incubation period, indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring waterper se.An orange suspension was observed; spectrophotometric analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 3.089, indicating that the test item has a potential interfering ability. Based on these results, additional controls were 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 0.38 cm2(treatment level: 53 mg/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco's phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSCliving) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. Since the test item is able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was performed.

In the Main Assay, the negative control gave 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], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

The positive control caused the expected cell death (4% of cell viability when compared to the negative control) and variability (SD of % viability equal to 1.8). Based on the stated criteria (mean viability < 40% and SD of % viability < 18), the assay was regarded as valid.