Alkali salt of sulfonated aryl azo amino sulfonyl aryl azo sulfonyl aryl azo aryl sulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2014 to 11 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reactive Brown DYHY 0331/0334

Test animals

Details on test animals or test system and environmental conditions:

Test System: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69007 Lyon –France).
Batch: 14-EKIN-028

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 minutes followed by a 42 ± 1 hour recovery period.

Number of animals:

Number of replicates: 3

Details on study design:

Positive control item: Sodium Dodecyl Sulphate (SDS) (SIGMA, batch 031M0035V), diluted at the final concentration of 5% (w/v) in sterile water
for injection (Baxter, batch 13A3106).

Negative control item: D-PBS (GIBCO, batch 1453554).

Non specific colour (NSC) control: test item treated tissues without MTT.

Non specific MTT reduction (NSMTT): killed tissues treated with the test item.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean cell viability

Run / experiment:

Time point: 42 h

Value:

116.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The Non Specific Colour (NSC), relative to the D-PBS Control, was 0.2%, lower than 5%. The non specific MTT reduction induced by the test item,
was 4.7% when compared to the negative control performed with alive tissues. Based on these results, no correction of the % viability obtained for the alive test item treated tissues was performed.
The test item did not induce cell death in any replicate with a mean cell viability of 116.6% when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability = 14 lower than 18).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item should be considered not irritant.

Executive summary:

The potential of the test item Reactive Brown DYHY0331/0334 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 blue/purple coloured solution was observed at the end of the incubation period, indicating that the test item interacted directly with MTT. In a second step, the test item was assayed for the ability of colouring water per se; a dark blue coloured solution was observed, which indicates a colouring capacity of the test item. Based on these results, additional controls were added in the Main Assay. In 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 (5% of cell viability when compared to the negative control) and variability (SD of % viability equal to 2.3). 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 (both NSC and NSMTT < 5%), no correction of the % viability obtained for the alive treated tissues was performed.

The test item did not induce cell death in any replicate with a mean cell viability of 116.6% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 14 (lower than 18, as stated in the Study Protocol). Based on the results obtained, the test item Reactive Brown DYHY0331/0334 is classified as not irritant to the skin.