N'-(2-Cyano-5-methoxy-4-nitrophenyl)-N,N-dimethylmethanimid amide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th August 2011 to 2nd November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Guideline:

other: European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N'-(2-Cyano-5-methoxy-4-nitrophenyl)-N,N-dimethylimidoformamide

EC Number:

813-618-8

Cas Number:

1269400-04-5

Molecular formula:

C11H12N4O3

IUPAC Name:

N'-(2-Cyano-5-methoxy-4-nitrophenyl)-N,N-dimethylimidoformamide

Test material form:

solid

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:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number: Lot no.:15619 kit N
-- Date of initiation of testing: 12 September 2011

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 - 37.4°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test substance

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 ml MTT solution (1 mg/ml in PBS).
- Incubation time: approximately 1 hour
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 4

PREDICTION MODEL / DECISION CRITERIA:
A test substance is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test substance considered non-corrosive (viability 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test substance is decreased below 15%.
A test substance is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Twenty-five mg of PF-05188294 was applied directly on top of the skin tissue. PF-05188294 was spread to match the size of the tissue.

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

Four

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour, mean

Value:

96

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute, mean

Value:

94

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

PF-05188294 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In vitro skin corrosion test with PF-05188294 using a human skin model.
This report describes the ability of PF-05188294 to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of PF-05188294 was tested through topical application for 3 minutes and 1 hour.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch E010011212 of PF-05188294 was an orange powder with a purity of 99.7%. Skin tissue was moistened with 25 μl of Milli-Q water and 25 mg of PF-05188294 was applied directly on top of the skin tissue.
The positive control had a mean relative tissue viability of 18% after 3 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues after 3 minutes exposure was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 24% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 14%, indicating that the test system functioned properly.
Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with PF-05188294 compared to the negative control tissues was 94% and 96%, respectively. Because the mean relative tissue viability for PF-05188294 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment PF-05188294 is considered to be not corrosive.
Finally, it is concluded that this test is valid and that PF-05188294 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.