Reaction product of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium chloride and Naphthalenesulfonic acids, reaction products with formaldehyde, ammonium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-09 to 2013-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with GLP regulations and OECD/EU guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

26 July 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tris(9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethyl-9H-xanthen-9-ylium) 6-[(3-sulfonatonaphthalen-2-yl)methyl]-3-[(6-sulfonatonaphthalen-2-yl)methyl]naphthalene-2-sulfonate

EC Number:

940-448-2

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

tris(9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethyl-9H-xanthen-9-ylium) 6-[(3-sulfonatonaphthalen-2-yl)methyl]-3-[(6-sulfonatonaphthalen-2-yl)methyl]naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

other: EpiSkinSM

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM
EpiSkinSM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
- Supplier: SKINETHIC Laboratories; 4, rue Alexandre Fleming, 69007 – LYON, France.
- Batch No.: 13-EKIN-035
- Expiry date: 14 October 2013

Test system

Type of coverage:

other: EpiSkin model

Preparation of test site:

other: EpiSkin model

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 10 mg of the test item

Duration of treatment / exposure:

15 min

Observation period:

42 hours

Number of animals:

3 replicates per test item
3 replicates negative controls
3 replicates positive controls
3 replicates of additional control for coloured test item (i.e. for non-specific OD evaluation)

Details on study design:

TEST SITE
- Area of exposure: the whole surface of the EpiSkin model

REMOVAL OF TEST SUBSTANCE
- Washing: PBS 1x solution
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Viability of the test item treated EpiSkin model in comparison to negative control.
The test substance is considered to be a skin irritant (R38), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Viability [% control]

Value:

87

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 42 h. Max. score: 100.0. Remarks: Test item. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Viability [% control]

Value:

26

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 42 h. Max. score: 100.0. Remarks: Positive control (SDS 5% aq.). (migrated information)

In vivo

Irritant / corrosive response data:

- Cell viability
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.

Other effects:

- Validity of the test
The mean OD value of the three negative control tissues was 0.862. The mean OD value obtained for the positive control was 0.224 and this result corresponds to 26 % viability when compared to the results obtained from the negative controls. 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 substance:
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 substances:
As the test item has an intrinsic colour (red), three additional chemical-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues were determined as 0.025. The Non Specific Colour% (NSC %) was calculated as 2.9 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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. According to the current OECD Guideline No. 439, DOCU Red 116 is considered as non-irritant to skin and is therefore not classified.

Executive summary:

The purpose of the study was to determine the skin irritation potential of the test item DOCU Red 116 on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

SDS 5 % aq. and 1 x PBS treated epidermis units 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 is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50% when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item DOCU Red 116 did not show significantly reduced cell viability in comparison to the negative control. 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.

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. According to the current OECD Guideline No. 439, DOCU Red 116 is considered as non-irritant to skin and is therefore not classified.