Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 18 February 2014; Experimental Completion Date: 24 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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)

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:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

EC Number:

289-056-7

EC Name:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Cas Number:

85959-61-1

Molecular formula:

C162H180N12O18Cl6+O3.P2O5.xWO3.yMoO3

IUPAC Name:

heptakis(N-[(3E)-6-(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-3H-xanthen-3-ylidene]ethan-1-aminium) dioxomolybdenumbis(olate) dioxotungstenbis(olate) phosphate

Test material form:

other: granula solid

Details on test material:

Identification: Pigement Red 81:4
Batch: S-3101
Physical state / Appearance: magenta colored granula solid
Expiry Date: 01 June 2014
Storage Conditions: Room temperature in the dark

Test animals

Species:

other: Not applicable. EPISKIN Reconstructed Human Epidermis (RHE) Model used.

Strain:

other: Not applicable.

Details on test animals or test system and environmental conditions:

Details on test animals not applicable for in-vitro study.

EPISKIN Reconstructed Human Epidermis Model Kit supplied by SkinEthic Laboratories, Lyon, France

Test system

Type of coverage:

other: Not applicable.

Preparation of test site:

other: Not applicable.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

10 mg of the test item was then applied to the epidermal surface.

Duration of treatment / exposure:

Tissues were treated with the test item for an exposure period of 15 minutes.

Observation period:

42 hours post exposure incubation period.

Number of animals:

Not applicable.

Details on study design:

TEST ITEM FORMULATION AND EXPERIMENTAL PREPARATION:
The test item was used as supplied.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS, MTT AND ACIDIFIED ISOPROPANOL:
The negative control item, PBS, was used as supplied.
The positive control item, SDS, was prepared as a 5% w/v aqueous solution.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Negative Control:
Phosphate Buffered Saline Dulbecco’s (PBS) with Ca++ and Mg++

Positive Control:
Sodium Dodecyl Sulphate (SDS).

PRE-TEST PROCEDURE
ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT:
MTT Salt Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:

10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL):
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:

Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes

2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

MAIN TEST:
APPLICATION OF TEST ITEM AND RINSING (DAY 1):
2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12 well plate.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve further contact between the test item and the epidermis. 10 mg of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7 Minutes contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.

MTT LOADING/FORMAZAN EXTRACTION (DAY 3):
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.

2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.

For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Relative mean viability (%)

Value:

89.7

Remarks on result:

other:

Remarks:

Basis: mean (test item). Time point: 15-Minute exposure period and 42 hours post-exposure incubation period.. Remarks: Non-irritant. (migrated information)

In vivo

Irritant / corrosive response data:

The relative mean viability ofthe test item treated tissues was 89.7% after a 15-Minute exposure period and 42 hours post-exposure incubation period.

It was considered unnecessary to perform IL-lα analysis as the results of the MTT test were unequivocal.

Other effects:

Direct MTT Reduction:
The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Any other information on results incl. tables

The individual and mean OD₅₆₂ values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in the table below. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in the table.

Mean OD₅₆₂ Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.870	0.988	0.102	88.1	100*	10.3
	1.046			105.9
	1.047			106.0
Positive Control Item	0.043	0.046	0.004	4.4	4.7	0.4
	0.050			5.1
	0.045			4.6
Test Item	0.899	0.887	0.047	91.0	89.7	4.7
	0.926			93.7
	0.835			84.5

* The mean viability of the negative control tissues is set at 100%.

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was 4.7% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.4%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₆₂ for the negative control treated tissues was 0.988 and the standard deviation value of the percentage viability was 10.3%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 4.7%. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information (non-irritating) Criteria used for interpretation of results: EU

Conclusions:

The test item was classified as non-irritant.