Disodium 3-[[2,2'-dimethyl-4'-[[4-[[(p-tolyl)sulphonyl]oxy]phenyl]azo][1,1'-biphenyl]-4-yl]azo]-4-hydroxynaphthalene-2,7-disulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 19th to June 21th, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

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)

Version / remarks:

2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acid Red 111

IUPAC Name:

Acid Red 111

In vitro test system

Test system:

isolated skin discs

Source species:

human

Cell type:

other: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purpose of distinguishing between skin irritating and no-skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

-Pre-incubation: the "maintenance medium" was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 ml per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37±1°C in an incubator with 5±1 % CO2, >/=95 % humidified atmosphere.

-Application: three replicates were used for the test item and positive and negative controls respectively. Test item: the epidermal surface was first moistened with 5 µl deionised water in order to improve further contact between powder and epidermis. Subsequently, approximately 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necesssary. Positive and negative control: a volume of 10 µl positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette trip in order to cover evenly all the epidermal surface if necessary. Additional controls for dyes and chemicals able to colour the tissue: in addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

-Exposure: the plates with the treated epidermis units were incubated for the exposure time of 15 minutes (±0.5 min) at room temperature.

-Rinsing: after the incubation time, the EpiSkin SM units were removed and rinsed thoroughly with approximately 25 ml PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

-Post-incubation: after rinsing the units were placed into the plate wells with fresh pre-warmed "maintenance medium" (2ml/well) below them and then incubated for 42 hours (±1 h) at 37±1°C in an incubator with 5±1 % CO2 , >/= 95 % humidified atmosphere.

-MTT test after 42 hours incubation: after the 42 hours (±1 h) incubation, the EpiSkin Sm units were transferred into the MTT solution filled wells ( 2 ml of 0.3 mg/ml MTT per well) and then incubated for 3 hours (± 5 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, >/=95 % himidified atmosphere.

-Formazan extraction: at the end of incubation with MTT a formazan extraction was undertaken: a disk of epidermis was cut from the units (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µl acidified isopropanol (one tube corresponding to one well of the tissue culture plate). The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for approximately four hours at room temperature protected from light with gentle agitation (ca. 150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

-Cell viability measurements: following the formazan extraction, 2x200 µl sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance/Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µl).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 minutes ±0.5 min

Duration of post-treatment incubation (if applicable):

Epidermis units were incubated for 42 hours (±1 h)

Number of replicates:

three

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 1

Value:

92

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 2

Value:

97

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 3

Value:

93

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean OD value of the three negative controls tissues was 0.951. The mean OD value obtained for the positive control was 0.204 and this result corresponds to 21 % viability when compared to the reults obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Possible direct MTT reduction with test item: no colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore addittional controls and data calculations were not necessary. A false estimation of viability can be precluded.
Colouring potential of test item: the test item has an intrinsic colour red. If the test item is not white, off white, almost white and/or colorless the addtional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents the false viability, which can possibly be caused by the stained surface of the tissues by the test item. Two addtional test item-treated tissues were used for the non.specific OD evaluation. The mean OD (measured at 570 nm) of these tissues was determined as 0.060. The NSCliving % was calculated as 6.3 %. Therefore, additional data calculation was necessary.

Any other information on results incl. tables

OD values and viability percentages of the controls

Substance	Optical Density (OD)		Viability (%)
Negative Control:	1	1.002	105
1x PBS	2	0.899	94
	3	0.952	100
	mean	0.951	100
	standard deviation (SD)		5.46
Positive Control:	1	0.238	25
SDS (5 % aq.)	2	0.197	21
	3	0.178	19
	mean	0.204	21
	standard deviation (SD)		3.2

OD values and viability percentages of the test item (including corrected values)

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Acid Red 111	1	0.871	0.811	92	85
	2	0.921	0.861	97	90
	3	0.885	0.824	93	87
	mean	0.892	0.832	94	87
	standard deviation (SD)			2.68	2.68

TOD_TT: OD true MTT metabolic conversion for treated tissues

OD values and NSC_living% of additional control

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSCliving %)
Acid Red 111 (test item treated tissues without MTT incubation)	1	0.052	6.3
	2	0.068
	mean	0.06

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (</=) to 50 % of the negative control.

Applicant's summary and conclusion

Interpretation of results:

other: According to CLP Regulation (EC) 1272/2008 criteria the test item do not need to be classified.

Conclusions:

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean corrected value:87 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non.irritant to skin. Positive and negative controls showed the expected ODs, cell viability values were within acceptable limits and standard deviations of all calculated viability values (positive and negative control, test item) were below 18. The experiment was considered to be valid.
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilisied testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified.
According to CLP Regulation (EC) 1272/2008 criteria the test item do not need to be classified.

Executive summary:

EpiSkin SM test of the test item has been performed to predict its irritation potential by measurements of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes (±0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (±1 h) in an incubator with 5±1 % CO2, >/= 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1°C in 5±1 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item has an intrisic colour (red), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

SDS (5 % aq.) and 1x PBS treated (three units/positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test chemical is identified as requiring classification and labelling according to UN GHS (category 2 or category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (</=) to 50 % of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean corrected value: 87 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected ODs, cell viability values were within acceptable limits and standars deviations of all calculated viability values (positive and negative control, test item) were below 18. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified.