Tetrakis{9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium} C12-(branched and/or linear)-alkyl-(4-sulfonatophenoxy)benzenesulfonate and oxybis[C12-(branched and/or linear)-alkyl-benzenesulfonate]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reliability is rated 2 because this is the result of a read-across from an analogue of the same category of substance, namely the organic dyes synthetized from a cationic and an anionic dyes. The reliability of the crossed-reference substance’s results is rated 1 because the study is equivalent of the standard guideline OECD 437. The results were reviewed and assessed as valid, and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 437

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sepisol Fast Violet 3B

IUPAC Name:

Sepisol Fast Violet 3B

Constituent 2

Reference substance name:

Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

IUPAC Name:

Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

Constituent 3

Reference substance name:

700-615-0

EC Number:

700-615-0

IUPAC Name:

700-615-0

Details on test material:

- Name of test material (as cited in study report): Sepisol Fast Violet 3B
- Substance type: Organocopper
- Physical state: Dark violet Powder
- Lot/batch No.: 028942
- Stability under test conditions: Stable
- Storage condition of test material: room temperature

Test animals / tissue source

Species:

other: isolated corneas from the eyes of freshly slaughtered cattle

Details on test animals or tissues and environmental conditions:

TEST EYES
- Source: Slaughterhouses Sobeval Boulazac 24759 - France
- Age of the donors animals: less than 8 month
- Transport of eyes: Balanced salt solution
- Time between collection and use of corneas:The corneas were transported to the laboratory straight after removal on animals and used in a maximum delay of 24 hours after reception at the laboratory.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

750 mg+/-75 mg

Duration of treatment / exposure:

30 min and 10 min.

Number of animals or in vitro replicates:

3 eyes for the negative control corneas and 3 eyes for the treatment group for each contact times (30 et 10 minutes).

Details on study design:

Selection of corneas free of defects.

They were dissected with a 2 to 3 mm rim of sclera to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium and endothelium. Isolated corneas were mounted on a corneal holder which was composed of an anterior and a posterior compartment which interfaced with the epithelial and endothelial sides of the cornea, respectively.

Both compartments were filled with a nutritious medium.

After a preincubation period (at 32 +/-1°C for at least one hour in a water bath), the baseline opacity of each cornea was determined.
Then the posterior compartment was filled with the nutrious medium. In the anterior compartment, 750 mg +/- 75 mg of the substance test was applied by direct application onto the corneal surface, in order to cover the entire corneal surface. For the negative control, the anterior compartment was filled with 750 µL of the nutrious medium.

Corneal holders were incubated in a water bath (32°C) for 30 (first assay) or 10 min (second assay).

After exposure period, the test substance is removed from the anterior chamber (a cotton bud was necessary to remove the test item in excess) and epithelium was washed at least three times. The anterior compartment was refilled with the nutrient medium and corneas were incubated for an additional two hours at 32 +/-1°C in a wash bath.

The opacity and permeability of each cornea were recorded:
- The opacitometer was calibrated before each readings: one before the application of the test item and a second one two hours after the end of exposure.
- The permeability was determined after the second reading of opacity by optical density (VISIONLite 2.2 v software). 1 ml of fluorescein solution was added to the anterior compartment and the holder was then incubated in a horizontal position for 90 +/-5 min at 32 +/-1°C.

At the end of the study, each cornea was visually checked and observations were recorded.

The in vitro irritancy score (IVIS) for each treatment group was calculated as follows:
IVIS= mean corrected opacity value + (15 x mean corrected permeability optical density)

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: In vitro Irritancy Score (IVIS)

Basis:

mean

Time point:

other: 30 min contact time

Score:

11.2

Results 2

Irritation parameter:

other: In vitro Irritancy Score (IVIS)

Basis:

mean

Time point:

other: 10 min contact time

Score:

0.9

Irritant / corrosive response data:

According to the rating scale, Sepisol Fast Violet 3B tested as such by direct application was found to be moderately irritant.
Nonetheless, corneas from the treatment group were colored in blue after 30 min of contact. Since opacity can be due to the corneal blue coloration and not due to a real corneal opacity, the result in uncertain.

Other effects:

After a contact of 30 min with the test item, the cornea was colored in blue with an epithelial detachment.

Any other information on results incl. tables

Opacitometer calibration

		30 min contact time		10 min contact time
Opacity before Treatment (OPT 0)	Calibrator 1	-75	75	-75	75
	Calibrator 2	-153	150	-153	151
	Calibrator 3	-230	225	-230	227
Opacity 2h after Rinsing (OPT 2)	Calibrator 1	-74	75	-76	75
	Calibrator 2	-152	152	-153	152
	Calibrator 3	-229	227	-235	229

OPT 0: Opacity 0 before treatment

OPT 2: Opacity 2h after rinsing

cOPT: corrected Opacity

cOD: corrected Optical Density

In vitroIrritancy Score (IVIS)

		30 min contact time
		OPACITY				PERMEABILITY
Holder N°	Treatment	OPT 0	OPT 2	OPT 0-OPT 2	cOPT	OD	cOD	IVSI
15	Negative control	0	0	0		0.019	0.019	0.3
2		2	2	0		0.029	0.029	0.4
12		1	0	-1		0.029	0.029	0.4
MOYENNE (écart-type)					-0.3 (0.6)	0.026 (0.006)		0.4 (0.1)
13	Sepisol Fast Violet 3B	5	12	7	7.3	0.168	0.142	9.5
8		0	11	11	11.3	0.081	0.055	12.2
56		1	10	9	9.3	0.201	0.175	12.0
MOYENNE (écart-type)					9.3 (2.0)	0.124 (0.062)		11.2 (1.5)

Control OD (fluorescein solution at 5 µg/ml) = 0.863

		10 min contact time
		OPACITY				PERMEABILITY
Holder N°	Treatment	OPT 0	OPT 2	OPT 0-OPT 2	cOPT	OD	cOD	IVSI
3	Negative control	0	2	2		0.022	0.022	0.3
14		0	0	0		0.019	0.019	0.3
44		-1	1	2		0.013	0.013	0.2
MOYENNE (écart-type)					1.3 (1.2)	0.018 (0.005)		0.3 (0.1)
48	Sepisol Fast Violet 3B	2	2	0	-1.3	0.080	0.062	-0.4
1		0	3	3	1.7	0.109	0.091	3.0
2		0	1	1	-0.3	0.049	0.031	0.1
MOYENNE (écart-type)					0.0 (1.5)	0.061 (0.030)		0.9 (1.8)

Control OD (fluorescein solution at 5 µg/ml) = 0.871

Applicant's summary and conclusion

Interpretation of results:

moderately irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: Gautheron0 P. et al - Fundam. Appl. Toxicol., 1992, 18, 442-449.

Conclusions:

The test item is considered moderately irritating with uncertainty.

Executive summary:

The aim of the study was to assess quantitatively the irritant potential of Sepisol Fast Violet 3B after the application to the isolated calf cornea. The assessment was based on the measurement of two parameters: the opacity and permeability of the cornea whose deteriorations reflected the damage of the tissue. The test element was let in contact with the isolated cornea for 30 minutes (first assay) and 10 minutes (second assay).

On the basis of the results obtained for each time of contact, a corneal score was calculated and the test element classified moderately irritant. However, corneas from the treatment group were colored in blue after 30 min of contact. Since opacity can be due to the corneal blue coloration and not due to a real corneal opacity, the result is uncertain. Furthermore, the laboratory doesn't have any recoil on this category of substances (i.e. organic dye) and no comparison with an appropriate database was performed. So the accuracy of BCOP and its applicability domain for this category of substance cannot be assessed and the results are uncertain.

Those identified limitations for the BCOP assay (i.e. coloration of the test material and uncertainty of the applicability) can be extrapolated to the other in vitro tests method (validated or not), since (1) their current validation database did not allow an adequate evaluation of organic dyes class and (2) the coloring property of the dye is most likely to interfere with the implementation of the test protocols and/or the interpretation of the results. Therefore, the realization of an in vitro assay, to test the eye irritancy potency of the Sepisol Fast Red SN is not technically feasible.