Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 November 2014 to 16 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; coherence between data, results and conclusion.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test item: Disperse Blue CVG

Test animals

Details on test animals and environmental conditions:
Test system: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69007 Lyon – France).
Batch numbers: 15-EKIN-006 (alive tissue) and 15-EKIN-005 (killed tissues)

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2).
Duration of treatment / exposure:
Exposure period of 15 ± 0.5 minutes followed by a 42 ± 1 hour recovery period.
Number of animals:
Number of replicates: 3
Details on study design:
Positive control item was sodium dodecyl sulphate (SDS) (SIGMA, batch 041M8713V), diluted at the final concentration of 5% (w/v) in sterile water for injection (Baxter, batch 13L0503).
Negative control item was D-PBS (GIBCO, batch 1605086).
Non specific colour (NSC) control: test item treated tissues without MTT.
Non specific MTT reduction (NSMTT) control: killed tissues treated with the test item.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: mean relative viability
Value:
105
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 h. Max. score: 50.0. Remarks: colorimetric measurement of MTT reduction; Mean relative viability <=50%: Irritant (UNGHS Category 2) Mean relative viability > 50%: Not irritant (UNGHS No Category). (migrated information)

In vivo

Irritant / corrosive response data:
The Non Specific Colour (NSC), relative to the negative control, was 8.3%, while the non specific MTT reduction (NSMTT) induced by the test item was 12.2%. These results indicated the necessity of additional background subtractions for the evaluation of irritant properties of Disperse Blue CVG.
Following the appropriate subtractions, a mean cell viability of 105%, when compared to the negative control, was obtained with the test item treated tissues. Intra-replicate variability was acceptable with a SD of % viability value equal to 1.4 (lower than 18, as stated in the Study Protocol).

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The test item should be considered not irritant.
Executive summary:

The potential of the test item Disperse Blue CVG to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. 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; a green coloured suspension with green precipitate was observed at the end of the incubation period, indicating that the test item could interact directly with MTT. In a second step, the test item was assayed for the ability of colouring water per se; a green suspension was observed, which indicates a colouring capacity of the test item. 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 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. 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 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 (4.2% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.8). Based on the stated criteria (mean viability 40% and SD of % viability 18 ), the assay was regarded as valid.

In order to verify if the test item results had to be corrected, the non specific colour (NSC) was evaluated using one alive treated tissue without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using killed tissues and compared with negative control performed with alive tissues. Based on the results obtained (NSC=8.3% and NSMTT=12.2%), additional background subtractions were performed for the evaluation of irritant properties of Disperse Blue CVG. The test item did not induce cell death in any replicate with a mean cell viability of 105% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 1.4 (lower than 18, as stated in the Study Protocol). Based on the results obtained, the test item Disperse Blue CVG is classified as not irritant to the skin.