Sodium 5-methyl-N-[2-(4-sulphamoylphenyl)ethyl]pyrazinecarboxamidate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-06-06 to 2017-06-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 5-methyl-N-[2-(4-sulphamoylphenyl)ethyl]pyrazinecarboxamidate

EC Number:

283-029-3

EC Name:

Sodium 5-methyl-N-[2-(4-sulphamoylphenyl)ethyl]pyrazinecarboxamidate

Cas Number:

84522-34-9

Molecular formula:

C14H16N4NaO3S

IUPAC Name:

5-methyl-N-[2-(4-sulfamoylphenyl)ethyl]pyrazine-2-carboxamide;sodium

Specific details on test material used for the study:

Batch: 16024R046
Purity: 100.3%

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 26424 Kit J
- Date of initiation of testing: 06 Jun 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubation of 3 minutes: at room temperature, others at 37.0 ± 1.0°C (actual range 36.5 - 37.6°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed with phosphate buffered saline, the rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: two tissues for 3-minute exposure and two for a 1-hour exposure, for the negative and positive controls, 2 tissues for both the 3-minute and 1-hour time point

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 26.0 to 42.6 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL 8N KOH

Duration of treatment / exposure:

3-minute and 1-hour

Number of replicates:

two tissues for 3-minute exposure and two for a 1-hour exposure, for the negative and positive controls, 2 tissues for both the 3-minute and 1-hour time point

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Value:

78

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The relative mean tissue viability obtained after the 3-minute treatments with the test item

Results 2

Irritation / corrosion parameter:

% tissue viability

Value:

7.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The relative mean tissue viability obtained after the 1-hour treatments with the test item

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by test item was -0.07% and -1.4% of the negative control tissues after 3 minutes and 1 hour respectively.
- Colour interference with MTT: a color change was observed by adding MTT-medium it was concluded that test item did interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range.
- Acceptance criteria met for positive control: The mean relative tissue viability following the 1-hour exposure to the positive control was 4.4%.
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤10%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The test item is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In vitro skin corrosion test was performed using a human skin model according to OECD 431 (2016).

 

Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 78% and 7.2%, respectively. Because the mean relative tissue viability for the test item was below 15% after the 1-hour treatment the test item is considered to be corrosive.

 

In conclusion, the test item is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.