4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, 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:

8.-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:

2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

EC Number:

287-722-1

EC Name:

4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Cas Number:

85567-10-8

Molecular formula:

C18H13N4Na3O12S3

IUPAC Name:

trisodium 5-oxo-4-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-1-(4-sulfonatophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate

Test material form:

solid

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:

unchanged (no vehicle)

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.
- Pre-incubation: 60 ± 5 minutes in the incubator (37 ± 1 °C, 5% CO2), then for about 18 ± 3 hours (37 ± 1 °C, 5% CO2).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg + 25 µL DPBS

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Test system

Details on study design:

Details of the test procedure used
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used
- Conditions of exposure: 37 ± 1 °C, 5% CO2
- Washing: inserts gently rinsed with DPBS
- Number of tissue replicates used per test chemical and controls: 3
- MTT assay: incubation with 0.3 mL of MTT solution for 60 minutes at 37 ± 1 °C, 5% CO2)

- 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%.

- Historical data positive control: Mean Viability:4.0%; Rel. Standard Deviation: 2.0%

- Historical data negative control: Mean Absorption: 1.830%; Rel. Standard Deviation:0.376%

- The test meets acceptance criteria if:
- mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Single test with three tissues

Value:

>= 94.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The acceptance criteria were met

Any other information on results incl. tables

Result of the Test Item

Name	NK			PC			TM
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	2.021	1.928	1.971	0.105	0.107	0.113	1.895	1.886	1.790
	2.020	1.959	2.045	0.114	0.110	0.118	1.888	1.932	1.888
OD570(blank-corrected)	1.978	1.885	1.928	0.062	0.064	0.070	1.852	1.843	1.747
	1.977	1.916	2.002	0.071	0.067	0.075	1.845	1.889	1.845
mean OD570of the duplicates (blank-corrected)	1.978	1.900	1.965	0.067	0.066	0.073	1.849	1.866	1.796
total mean OD570of 3 replicate tissues (blank-corrected)	1.948*			0.068			1.837
SD OD570	0.042			0.004			0.036
relative tissue viability [%]	101.5	97.6	100.9	3.4	3.4	3.7	94.9	95.8	92.2
mean relative tissue viability [%]	100.0			3.5**			94.3
SD tissue viability [%]***	2.1			0.2			1.9
CV [% viabilities]	2.1			5.5			2.0

 

^* 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%

NK negative control of living tissues

PC positive control

TM test item treated living tissues

Applicant's summary and conclusion

Interpretation of results:

other: the test item showed no irritant effects

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In this in vitro study the skin irritation potential of the test item Reactive Yellow 42 was assessed according to

OECD 439 Test Method and EU-Method B.46 and in compliance to GLP.

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.

In the present study Reactive Yellow 42 was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no non-specific reduction of MTT and no colouring potential after mixture with isopropanol. 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 > 50% (94.3%) after 60 min treatment and 42 h post-incubation.

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% - 2.1%).