Trisodium 5,5'-[(2-sulphonato-1,4-phenylene)bis(azo)]bis(salicylate)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-30 to 2017-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP 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:

other: EU Method B.46 (Skin Irritation)

Version / remarks:

July 2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 5,5'-[(2-sulphonato-1,4-phenylene)bis(azo)]bis(salicylate)

EC Number:

276-501-5

EC Name:

Trisodium 5,5'-[(2-sulphonato-1,4-phenylene)bis(azo)]bis(salicylate)

Cas Number:

72230-95-6

Molecular formula:

C20H14N4O9S.3Na

IUPAC Name:

trisodium 3,3'-[(2-sulfonato-1,4-phenylene)didiazene-2,1-diyl]bis(6-hydroxybenzoate)

In vitro test system

Test system:

human skin model

Justification for test system used:

Recommended in vitro model

Vehicle:

unchanged (no vehicle)

Remarks:

distilled water was applied to the epidermal surface in order to improve further contact between the powder and the epidermis

Details on test system:

Dose Groups
1. Negative control 10 µL DPBS
2. Positive control 10 µL 5% SDS solution
3. Test Item 10 ± 2 mg + 5 µL aqua dest.
The test was performed on a total of 3 tissues per dose group.
SDS sodium dodecyl sulfate
DPBS Dulbecco's phosphate buffered saline

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

10 ± 2 mg + 5 µL aqua dest.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 +/- 1 h

Number of replicates:

3

Test system

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, the negative control (10µL DPBS) 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 DPBS 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 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

96.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Test Acceptance Criteria
Value Cut off pass/fail
Mean OD570 nm Blank 0.043 < 0.1 pass
Mean Absolute OD570 nm NK 0.950 0.6 ≤ NK ≤1.5 pass
Mean Relative Viability [%] PC 17% ≤ 40% pass
SD of % Viability [%] 4.2% - 12.1% ≤ 18% pass

Any other information on results incl. tables

The mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. 

The mixtures of 10 mg of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed colouring detectable by unaided eye-assessment and the chemical in water and isopropanol absorbed light in the range of 570 ± 30 nm. Therefore, the non-specific colour (NSC_living) was determined and calculated according to the following formula:

NSC_living[%] = [OD_TVT/OD_NK]*100 = [0.0103/0.907]*100 = 1.1%

NSC_living was ≤ 5% (1.1%) relative to the negative control of living epidermis. No correction of results was necessary.

Result of the Test Item

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	0.931	0.905	0.990	0.108	0.157	0.308	0.857	0.892	0.977
	0.958	0.924	0.991	0.116	0.158	0.334	0.885	0.920	0.999
OD570(Blank-Corrected)	0.888	0.862	0.947	0.065	0.114	0.265	0.814	0.849	0.934
	0.915	0.881	0.948	0.073	0.115	0.291	0.842	0.877	0.956
Mean OD570Of The Duplicates (Blank-Corrected)	0.902	0.871	0.948	0.069	0.115	0.278	0.828	0.863	0.945
Total Mean OD570Of 3 Replicate Tissues (Blank-Corrected)	0.907*			0.154			0.878
SD OD570	0.038			0.110			0.060
Relative Tissue Viabilities [%]	99.4	96.1	104.5	7.6	12.6	30.7	91.3	95.1	104.2
Mean Relative Tissue Viability [%]	100.0			17.0**			96.9
SD Tissue Viability [%]^***	4.2			12.1			6.6
CV [% Viability]	4.2			71.6			6.8

^*        Corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

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

^***      Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

The mean tissue viabilities are calculated relatively to the mean of the negative controls.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Regulation (EC) no 1272/2008

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study the skin irritant potential of the test itemwas analysed. The EPISKIN-Standard Model™ (EPISKIN-SM^TM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404[8]). Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

The test item showed no non-specific MTT-reducing potential but it showed colouring potential after mixture with aqua dest. or isopropanol, which did not influence the validity of the study. NSC_living was determined to 1.1%, therefore no correction of results was necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (96.9%) after 15 min treatment and 42 h post-incubation. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.