2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2017 to 26 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

EC Number:

285-413-6

EC Name:

2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Cas Number:

85098-62-0

Molecular formula:

C30H15ClN7Na5O15S4

IUPAC Name:

2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Test material form:

solid: particulate/powder

Remarks:

Dark brown powder

Details on test material:

- Storage conditions: Controlled room temperature (15 - 25 °C, below 70 RH%) in an opaque plastic flask.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
> Living Tissues
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic, France.
- Tissue lot number: 17-EKIN-021
- Expiry date: 29 May 2017

> Killed Tissues
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic, France.
- Tissue lot number: 17-EKIN-003
- Expiry date: 23 January 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (24.9 - 26.8°C)
- Temperature of post-treatment incubation: 37°C

ASSESSMENT OF POSSIBLE MTT REDUCTION
10 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded.
After three hours incubation, purple red colour was detected in the test tube. Thus, the test material reacted with MTT and therefore the use of additional controls was necessary.

ASSESSMENT OF COLOURING POTENTIAL OF TEST MATERIAL
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or extracting solution. The test item was shown being an MTT-interacting substance and the test material had an intrinsic colour a third set of controls was necessary. To avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled) was needed.

Two additional test item-treated living tissues and two additional test item-treated killed tissues were used for the non-specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

MAIN STUDY

PRE-INCUBATION (DAY -1)
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was 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°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

APPLICATION OF TEST MATERIAL (DAY 0)
10 μL distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 10 mg of the test item was applied evenly to the epidermal
surface. The test material was spread gently on the skin surface with a pipette tip without damaging the epidermis.
50 μL of negative control or positive control were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly without damaging the epidermis.

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period, each tissue was removed from the well and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

MTT TEST (DAY 2)
After the 42 hours incubation, all tissues (except of two NSClving and two NSCkilled colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred tissues were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

FORMAZAN EXTRACTION (DAY 2)
After the incubation with MTT, a formazan extraction was undertaken. A disc of epidermis was cut from each skin unit (this involved 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 containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

NUMBER OF REPLICATE TISSUES: 3 (normal procedure) plus an additional two test material treated killed epidermis and two negative control treated killed epidermis was used in the study

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate at the required wavelength on each day before use.



Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-hour post-exposure incubation period according to:
- Relative mean tissue viability is ≤ 50%: Irritant (H315 Category 2)
- Relative mean tissue viability is > 50%: Non-irritant (Not classified for irritation)

-Quality criteria:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
- Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤ 40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤ 18%.
- Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is ≥ 0.6 and ≤ 1.5, and the SD value of the percentage viability is ≤ 18%.
- Test Material: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg wetted with 10 µL distilled water to improve contact

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 5% w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 (normal procedure) plus an additional two test material treated killed epidermis and two negative control treated killed epidermis was used in the study

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

63.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

As the test material was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissue was 0.014, Non-Specific Colour % was calculated as 2.0% (see Table 1). This value was below 5%, therefore additional data calculation was not necessary.

As colour change (purple red) was observed after three hours of incubation of the test material in MTT working solution, the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Results of the additional controls on killed epidermis are shown in Table 2. Based on the observed mean OD (-0.009), the calculated NSMTT% is -1.3%. Since the OD values were negative, therefore these values excluded from the NSMTT calculation.

As the test material was shown being an MTT-interacting substance and the test material had an intrinsic colour, two additional test material-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.021, Non-Specific Colour % (NSCkilled%) was calculated as 3.1% (see Table 3). Because the NSCliving% was not used, correction with NSCkilled% was not necessary.

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 4. The OD values for the test material treated skin samples showed 63.1% relative viability.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Executive summary:

An in vitro skin irritation test of the test material was performed in a reconstructed human epidermis model. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 439 and EU Method B.46.

During the study discs of EPISKIN^TM (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disc was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control).

Two additional discs were used to provide an estimate of colour contribution (NSCliving) from the test material. The possible MTT interaction potential of the test material was examined using two additional test item treated killed epidermis units and two negative control treated killed epidermis units. Furthermore to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. 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 item is considered to be irritant to skin.

Following exposure with test material, the mean cell viability was 63.1% compared to the negative control. This is above the threshold of 50%, therefore the test

material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.