Sodium 3-[{4-[{5-cyano-2,6-bis[(3-methoxyalkyl)amino]alkylpyridin-3-yl}diazenyl]dialkylphenyl}diazenyl]benzenesulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2015 to 29 June 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:

2015

Report date:

2015

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Test item: Acid Red EAY 9656

In vitro test system

Test system:

human skin model

Remarks:

reconstructed human epidermis

Source species:

human

Details on animal used as source of test system:

Test system: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69007 Lyon – France).
Batch numbers: Alive tissues: 15-EKIN-020 (Main Assay I), 15-EKIN-025 (Main Assay II).
Killed tissues: 15-EKIN-005 and 15-EKIN-006 (Main Assay I), 15-EKIN-020 (Main Assay II).

Vehicle:

unchanged (no vehicle)

Details on test system:

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.


Positive control item: Sodium Dodecyl Sulphate (SDS) (SIGMA, batch no. 041M8713V), diluted at the final concentration of 5% (v/v) in sterile water for injection (Baxter, batch nos.14C2402 and 14C2403).

Negative control item: D-PBS (GIBCO, batch no. 1605086).

Non Specific Colour (NSC) control: test item treated tissues without MTT.

Non Specific MTT Reduction (NSMTT): killed tissues treated with the test item.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

20 mg/epidermis unit, each measuring 0.38 cm² (treatment level: 53 mg/cm²)

Duration of treatment / exposure:

An exposure time of 15 +/- 0.5 minutes was allowed in a ventilated cabinet at room temperature. In Main Assay I, treatment was carried out staggering samples of approximately 1 minute, the exposure time for test item treated samples was approximately 30 minutes, due to the adhesion of the substance to the test system. In Main Assay II, test item treatment was carried out staggering samples of approximately 2 minutes while control tissues were staggered of 1 +/- 0.5 minutes.
Observation time was 42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean cell viability

Value:

86.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks on result:

other:

Other effects / acceptance of results:

In Main Assay I, the baseline value of the negative control was slightly lower than the acceptable one (mean OD value = 0.53313), hence a
second experiment (Main Assay II) was performed. However, result obtained for the non specific colour (NSC=3.0%) was considered reliable from Main Assay I for demonstrating the absence of colour interaction; this additional control was not repeated in Main Assay II.

In Main Assay II, the non specific MTT reduction (NSMTT) induced by the test item was 2.7%, thus only the OD-blank background subtraction was performed for the evaluation of irritant properties of Acid Red EAY 9656.

The test item did not induce cell death in any replicate with a mean cell viability of 86.6%, when compared to the negative control.
Acceptable intra-replicate variability was obtained (SD of % variability lower than 18).

Any other information on results incl. tables

Preliminary test

A reddish suspension was observed, with plentiful precipitate, which indicates a colouring capacity of the test item. For this reason, additional controls were added in the Main Assay for the evaluation of non specific colouring potential (NSC).

Main Assay I

The Non Specific Colour (NSC) was 3.0%, while the non specific MTT reduction (NSMTT) induced by the test item was 6.0%. Based on this result, appropriate background subtraction was carried out for the evaluation of test item mean cell viability. Using alive tissues, a mean cell viability of 89.0%, when compared to the negative control, was obtained after treatment with the test item.

Positive control results indicated appropriate relative cell viability (5.6 % of the negative control value) and variability between replicates (SD of % viability = 1.7). However, the baseline value of the negative control was slightly lower than the acceptable one (mean OD value = 0.53313), hence a second experiment was performed.

Since low values were obtained for the Non Specific Colour (NSC), results were considered reliable for demonstrating the absence of colour interaction and this additional control was not repeated in the second experiment. Relevant variability between replicates was noted in NSMTT determination, hence this additional control was repeated in Main Assay II.

 

Main Assay II

Using alive tissues, 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 negative control 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 (2.8 % of the negative control value). Variability between replicates gave also the expected value (SD of % viability = 0.1). 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 Non Specific Colour (NSC), relative to the negative control and obtained in Main Assay I, was 3.0%, while the non specific MTT reduction (NSMTT) induced by the test item was 2.7%. Based on these results, only the OD-blank background subtraction was performed for the evaluation of test item mean cell viability.

The test item did not induced any relevant reduction of MTT staining in any replicate. The mean cell viability was 86.6%, when compared to the negative control. Acceptable intrareplicate variability was obtained (SD of % viability = 5.2 lower than 18).

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

The test item Acid Red EAY 9656 is classified as not irritant to the skin.

Executive summary:

The potential of the test item Acid Red EAY 9656 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 Assays, 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 red coloured suspension, with dark red 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 reddish suspension, with plentiful precipitate, was observed, which indicates a colouring capacity of the test item. Based on these results, additional controls were added.

Two experiments were performed. In both experiments, 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% (v/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 (NSC) and the non specific MTT reduction (NSMTT) were also evaluated.

In Main Assay I, the negative control did not give the acceptable baseline value, thus a second experiment was performed. However, result obtained for the non specific colour (NSC=3.0%) was considered reliable for demonstrating the absence of colour interaction and this additional control was not repeated in the second experiment.

In Main Assay II, 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 (2.8% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.1). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18 ), the assay was regarded as valid. The non specific MTT reduction (NSMTT) induced by the test item was 2.7%, thus only the OD-blank background subtraction was performed for the evaluation of irritant properties of Acid Red EAY 9656.

The test item did not induce cell death in any replicate with a mean cell viability of 86.6%, when compared to the negative control. Based on the results obtained, the test item Acid Red EAY 9656 is classified as not irritant to the skin.