1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2016 to 27 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile

EC Number:

261-693-5

EC Name:

1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile

Cas Number:

59312-61-7

Molecular formula:

C20H16N4O5S

IUPAC Name:

3-[(5-cyano-2-hydroxy-1,4-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)diazenyl]phenyl benzenesulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Identification: FAT 93460/D
Physical state/Appearance: Yellow powder
Batch: 20140721 (China)
Purity: 94.6 %
Expiry Date: 14 October 2019
Storage Conditions: 4 °C in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen

Cell source:

other: EpiSkinTM Tissues (0.38 cm²) lot number 16-EKIN-025

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recognised in vitro test for skin irritation

Vehicle:

unchanged (no vehicle)

Details on test system:

Study Design:
Pre-Test Procedure:
Assessment of Direct Test Item Reduction of MTT:
MTT Salt Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results. A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 mg of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made. The test item was considered to have the potential to cause color interference with the MTT endpoint. Therefore, an additional procedure was performed using viable tissues to assess for the possibility of color interference. 10 mg of the test item was applied to three viable tissues in parallel to the main test with the exception that the MTT incubation period was replaced by incubation with maintenance medium (therefore without MTT). Three viable tissues were also used for negative control purposes which remained untreated. The optical density measurements from these tissues were then assessed for possible quantitative correction of the results.

Pre-incubation (Day 0: Tissue Arrival):
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes

2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5 % CO2 in air overnight.

Main Test:

Application of Test Item and Rinsing (Day 1):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm²) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5 % w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5 % CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3):
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg, used as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µL Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Sodium Dodecyl Sulphate (SDS)
- Concentration (if solution): 5 % w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 Minute exposure

Value:

83.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Mean OD₅₆₂ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	± SD of OD562	Mean OD562 of triplicate tissues	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.648			96.7
	0.611	0.670	0.072	91.2	100*	10.7
	0.750			111.9
Positive Control Item	0.099			14.8
	0.059	0.077	0.020	8.8	11.5	3.0
	0.074			11.0
Test Item	0.565			84.3
	0.558	0.561	0.004	83.3	83.7	0.6
	0.559			83.4

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

Executive summary:

A study was conducted in accordance with OECD test guideline 439 in a GLP-certified laboratory to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. 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. 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. The test item was shown to produce a colored solution therefore additional color correction tissues were included in parallel to the main test. 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). The relative mean viability of the test item treated tissues was 83.7 % after the 15-minute exposure period and 42-hours post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. In conclusion, the test item was classified as non-irritant. The following classification criteria apply: EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 cannot be determined).