2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-11-2016 to 25-11-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

28 July 2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

EC Number:

287-719-5

EC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Cas Number:

85567-07-3

Molecular formula:

C36H42Cl2KN6NaO28S8

IUPAC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Test material form:

solid

Details on test material:

Batch: 1283067001

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

other: not applicable

Vehicle:

other: DPBS to improve the contact between the powder and the epidermis

Details on test system:

- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
- Surface: 0.63 cm.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- test item: 25 mg + 25 µL DPBS (Dulbecco's phosphate buffered saline solution)
- negative and positive controls 30 µL each

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

3 per dose group

Test system

Details on study design:

- Pre-incubation: 18 ± 3 h in a humidified incubator at 37 ± 1 °C, 5% CO2
- Conditions of exposure: 37 ± 1 °C, 5% CO2
- Washing: inserts gently rinsed with DPBS
- MTT assay: incubation with 0.3 mL of MTT solution for 3 h ± 5 min at 37 ± 1 °C

Data evaluation:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”)], if the tissue viability after exposure and post-incubation is less or equal to 50%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Single test with three tissues

Value:

89.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The controls confirmed the validity of the study.
- the mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8
- the mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.5%)
- standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% - 10.3%)

Any other information on results incl. tables

Results:

Name	Negative control			Positive control			test material
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	2.021	1.928	1.971	0.105	0.107	0.113	1.607	1.712	1.989
	2.020	1.959	2.045	0.114	0.110	0.118	1.647	1.749	2.039
OD570(blank-corrected)	1.978	1.885	1.928	0.062	0.064	0.070	1.564	1.669	1.946
	1.977	1.916	2.002	0.071	0.067	0.075	1.604	1.706	1.996
mean OD570of the duplicates (blank-corrected)	1.978	1.900	1.965	0.067	0.066	0.073	1.584	1.687	1.971
total mean OD570of 3 replicate tissues (blank-corrected)	1.948*			0.068			1.748
St. Dev. OD570	0.042			0.004			0.201
relative tissue viability [%]	101.5	97.6	100.9	3.4	3.4	3.7	81.3	86.6	101.2
mean relative tissue viability [%]	100.0			3.5**			89.7
St. Dev. tissue viability [%]***	2.1			0.2			10.3
CV [% viabilities]	2.1			5.5			11.5

* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is ≤ 20%.

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects.

Executive summary:

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm (MatTek) comprising a reconstructed epidermis with a functional stratum corneum. The test was performed according to OECD TG 439 and in compliance to GLP.

The test substance was applied topically to the EpiDerm tissue. After a 60 min exposure and a 42 h post-incubation period, the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT was compared to those of the concurrent negative controls. No non-specific reduction of MTT and no colouring potential after mixture with isopropanol was observed. The mixture of the test item with aqua dest. showed colouring in the relevant wavelength range, therefore NSC_livingwas determined. Since NSC_livingwas ≤ 5% (0.4%), no correction of results was necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was 89.7 % after 60 min treatment and 42 h post-incubation, which is above the cut-off value of 50%.

The controls confirmed the validity of the study. The mean absolute OD₅₇₀of the three negative control tissues was ≥ 0.8 and ≤ 2.8. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.5%). Standard deviation of viability of replicate tissues of all dose groups was≤ 18% (0.2% - 10.3%).

Under the given conditions the test substance showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS and CLP.