Reaction mass of isomers disodium 8-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-5-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate, disodium 5-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-8-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-09 to 2013-04-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test material

Test material information

1

Reference substance name:

tetrasodium 5-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-8-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate 8-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-5-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

EC Number:

700-946-0

Molecular formula:

C40H35N7Na2O6S2

IUPAC Name:

tetrasodium 5-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-8-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate 8-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-5-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Test system

Amount / concentration applied:

10 mg + 15 µL aqua dest

Duration of treatment / exposure:

15 minutes

Observation period:

42 +/- 1 h

Details on study design:

The test was performed on EpiSkin, an organotypic reconstructed three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test item (10 mg + 15 µL A. dest. to ensure good contact and even distribution), the negative control (10µL PBS) and the positive control (10µL 5% SDS), respectively. After 15 minutes treatment period at room temperature the test item and the controls are rinsed off with PBS and the tissues are post-incubated for 42 +/- 1 h. Then the tissues are stained via MTT for 3 hours. Isopropanol extracts are measured photometrically at 550 nm.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

86.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

89.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item showed colouring of MTT medium in the pre-experiment. The mixture became dark purple to black. It could not be judged distinctly if the colourization was due to reduction of MTT or due to the strong intrinsic colour of the test item. Therefore, the part of absorption due to the possible non specific reduction of MTT (NSMTT = 18.4%) was determined by using killed tissues for quantitative correction of results to get the worst-case results.
The test item also showed colouring potential in the pre-experiment. Since the test item was not extractable from the tissues, no additional viable tissue controls were performed.

Any other information on results incl. tables

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2
OD550(TODTT)	1.056	0.935	1.002	0.134	0.092	0.104	0.867	0.898
	1.002	0.872	0.963	0.130	0.091	0.099	0.830	0.856
blank-corrected OD550(TODTT)	1.014	0.894	0.960	0.092	0.050	0.062	0.825	0.856
	0.960	0.830	0.922	0.088	0.049	0.057	0.788	0.814
blank-corrected mean OD550of the duplicates (TODTT)	0.987	0.862	0.941	0.090	0.050	0.060	0.807	0.835
blank-corrected total mean OD550of replicate tissues (TODTT)	0.930*			0.066			0.821
SD OD550(TODTT)	0.064			0.019			0.028
NSMTT-corrected relative tissue viabilities [%]	106.2	92.7	101.2	9.7	5.4	6.4	86.8	89.8
NSMTT-corrected mean relative tissue viability [%]	100.0			7.1**			88.3
SD NSMTT-correctedtissue viability [%]^***	6.8			2.3			2.2
CV [% viability]	6.8			31.5			2.5

*            Corrected mean OD550 of the negative control corresponds to 100% absolute tissue viability.

**         Mean relative tissue viability of the three positive control tissues is ≤ 40%.

***       The standard deviation (SD) obtained from the three concurrently tested tissues is < 18%. Only two tissues treated with the test item were evaluable.

Applicant's summary and conclusion

Interpretation of results:

other: not classified for skin irritation according to CLP criteria

Conclusions:

The test item is classified as "non-irritant".

Executive summary:

In the in vitro skin irritation test using the EpiSkin human epidermis model 10 mg test item + 15 µL A. dest. were applied topically for 15 minutes. After 42 h post-incubation cytotoxic effects were determined via MTT reduction assay.

The test item showed purple/black colouring in concact with MTT medium. Therefore, killed tissues were treated with the test item to determine Non-Specific MTT reduction.

 

The NSMT-corrected mean relative tissue viability (% negative control) was > 50%.

The test item is therefore classified as "non-irritant" (EU CLP and UN GHS: No Category).

 

This study is classified as acceptable:

OD₅₅₀ of the blank is < 0.1.

Mean OD₅₅₀ of the three negative control tissues is >= 0.6 and <= 1.5.

Mean relative tissue viability of the three positive contol tissues is <= 40%.

Standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is < 18%.

 

This study satisfies the requirement for Test Guideline OECD 439 for in vitro skin irritation.