2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

February from 01 to 03, 2017

Reliability:

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

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acid Yelow 236

IUPAC Name:

Acid Yelow 236

Specific details on test material used for the study:

The test item was applied in its original form, no formulation was required.

In vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

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 irritation, 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 purposes of distinguishing between skin irritating and non-skin irritating test substances.

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model, three-dimensional human epidermis model.
- Source: EPISKIN SNC Lyon, France.
- Tissue batch number: 17-EKIN-005
- Seeding: adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Culturing: a highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Demonstration of proficiency: prior to routine use of the method, the testing laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.
- Health check: all biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

CONDITIONS USED FOR TEST SYSTEM
- Conditions used during treatment / exposure: 37 °C in an incubator with 5 % CO2, ≥ 95 % humidified atmosphere.
- Conditions of post-treatment incubation: 37 °C in an incubator with 5 % CO2, ≥ 95 % humidified atmosphere.

PRE-INCUBATION, day [-1]-0
- Filling volume: the appropriate number of an assay plate wells were filled with the pre-warmed medium, 2 ml per well.
- Incubation: the epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight, 18 - 24hours.

APPLICATION, day 0
- Pre-treatment: the epidermal surface was first moistened with 5 µl deionised water in order to improve further contact between powder and epidermis.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing thoroughly with approximately 25 ml PBS 1x solution. 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, day 0-2
- Incubation after rinsing: units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 ml/well).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT stock solution: dissolved to a final concentration of 3 mg/ml in saline buffer. The obtained stock solution can be stored in refrigerator (2-8 °C), protected from light up to 15 days.
- MTT concentration: approximately 10 mg test item was added to 2 mL MTT 0.3 mg/ml solution and mixed.
- Incubation time: incubation for three hours at 37 °C protected from light.
- Plate: 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.
- Duration of incubation: approximately four hours.
- Conditions of incubation: room temperature protected from light with gentle agitation (~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, day 2
Following the formazan extraction, 2×200 µl sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at 570 nm (± 10 nm; Read out range: 0-3.5 Abs) using acidified isopropanol solution as the blank (6×200 µl).

ACCEPTANCE CRITERIA
- The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.
- for test chemicals, the standard deviation value (SD) of the % viability between tissue replicates should be ≤ 18.

PREDICTION MODEL / DECISION CRITERIA
- 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.
- The test substance may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg, 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 necessary.

NEGATIVE CONTROL
- Amount applied: 10 µl applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.

POSITIVE CONTROL
- Amount applied: 10 µl applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Duration of treatment / exposure:

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

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

Test item: 3 replicates
Positive and negative controls: 3 replicates
Colour control: 2 replicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

106

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 OD values obtained for the three replicates of the test item were 0.752, 0.979 and 0.755, corresponds to 96, 125 and 96 % viability, when compared to the results obtained from the negative control.

ACCEPTANCE CRITERIA
The mean OD value of the three negative control tissues was 0.786.
The mean OD value obtained for the positive control was 0.163 and this result corresponds to 21 % viability when compared to the results 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.

INDICATOR FOR POTENTIAL FALSE VIABILITY
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 additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test item: mean OD (measured at 570 nm) of these tissues was determined as 0.018. The Non Specific Colour % (NSC %) was calculated as 2.3 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Any other information on results incl. tables

OD values and viability percentages of test item

Substance	Optical Density (OD)		Viability (%)
Test item	1	0.752	96
	2	0.979	125
	3	0.755	96
	mean	0.829	106
	standard deviation (SD)		16.57

OD values and viability percentages of the controls

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.812	103
	2	0.721	92
	3	0.824	105
	mean	0.786	100
	standard deviation (SD)		7.15
Positive Control: SDS (5 % aq.)	1	0.157	20
	2	0.177	23
	3	0.154	20
	mean	0.163	21
	standard deviation (SD)		1.59

OD values and NSC % of additional control

Additional colour control	Optical Density (OD)		Non Specific Colour % (NSC %)
Test item treated tissues without MTT incubation	1	0.02	2.3
	2	0.017
	mean	0.018

Applicant's summary and conclusion

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

The test item reveals no skin irritation potential under the utilised testing conditions.

Executive summary:

EpiSkinTM SM test has been performed to predict the irritation potential of the test item, by measurement 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 test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light, ≥ 95 % humidified atmosphere. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×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 item has an intrinsic colour, therefore two additional test item treated tissues were used for the non-specific OD evaluation. No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 106 %). All obtained test item viability results were 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 cell viability values within acceptable limits. The experiment was considered to be valid.

Conclusion

The test item reveals no skin irritation potential under the utilised testing conditions.