Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Deviations:

yes

Remarks:

See below

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See below

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

EC Number:

260-394-7

EC Name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

Cas Number:

56819-40-0

Molecular formula:

C32H21CrN10O11S.2Na

IUPAC Name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

Identification: FAT 20010/E
Batch: 59015
Purity: not supplied
Physical state / Appearance: Dark orange colored powder
Expiry date: 18 February 2020
Storage Conditions: room temperature in the dark

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN™ Reconstructed Human Epidermis Model

Source species:

human

Vehicle:

other: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 15-EKIN-028
- Delivery date: 14 July 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hr
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

NUMBER OF REPLICATE TISSUES: Triplicate

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2)

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Concentration (if solution): 10 μL of DPBS served

POSITIVE CONTROL
- Concentration (if solution): 10 μL of SDS (5% w/v)

Duration of treatment / exposure:

Test item: 15 minutes
Positive control SDS: 7 minutes.

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

Triplicate

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

62.8

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Direct MTT Reduction: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint: An assessment found that the colored test item may interfere with the MTT endpoint. Therefore, an additional procedure using viable color correction tissues was performed to measure any potential interference. However, the results obtained showed that negligible interference due to the color of the test item occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

The relative mean tissue viability for the positive control treated tissues was 12.1 % relative to the negative control treated tissues and the standard deviation value of the viability was 1.5%.
The positive control acceptance criterion was therefore satisfied.
The mean 0D562 for the negative control treated tissues was 0.783 and the standard deviation value of the viability was 6.2%. The negative control acceptance criterion was therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically treated test item tissues was 14.6%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD₅₆₂Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562of triplicate tissues	±SDof OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.828	0.783	0.049	105.7	100*	6.2
	0.731			93.4
	0.790			100.9
Positive Control Item	0.108	0.095	0.012	13.8	12.1	1.5
	0.086			11.0
	0.090			11.5
Test Item	0.367	0.492	0.114	46.9	62.8	14.6
	0.591			75.5
	0.517			66.0

SD = Standard Deviation

* = mean viability of the negative control tissues is set at 100 %

OD₅₆₂= optical density

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations of OECD Guideline for the Testing of Chemicals No. 439 and Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. An assessment found that the colored test item may interfere with the MTT endpoint. Therefore, an additional procedure using viable color correction tissues was performed to measure any potential interference. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Results

The relative mean viability of the test item treated tissues was 62.8 % after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period. An assessment found that the colored test item may interfere with the MTT endpoint. Therefore, an additional procedure using viable color correction tissues was performed to measure any potential interference. However, the results obtained showed that negligible interference due to the color of the test item occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

 

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

 

Conclusion:

The test item was classified as non-irritant according to EU DSD/CLP and UN GHS.