1-amino-N-[6-cyano-5-(trifluoromethyl)-3-pyridyl]cyclobutanecarboxamide

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 2016-01-11 to 2016-01-15

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

Version / remarks:

28 July 2015

Deviations:

yes

Remarks:

See rationale for reliability

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

31 May 2008

Deviations:

yes

Remarks:

See rationale for reliability

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide

Cas Number:

1950587-17-3

Molecular formula:

C12H11F3N4O

IUPAC Name:

1-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: JNJ-63632283-AAA (T003665)
- Physical state: solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: I15HD3050
- Expiration date of the lot/batch: 2017-08-17 (retest date)
- Appearance: White powder
- Purity: 98.1% (UHPLC determination with specification >=95%)
- Purity test date: 2015-10-29

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, the solid test item (27.25 to 40.01 mg) was applied neat directly on top of the skin tissue. The test item was spread to match the size of the tissue.

OTHER SPECIFICS
- Correction factor: 1.00
- pH (1% in water, indicative range): 6.8 – 6.7 (determined by WIL Research Europe)

In vitro test system

Test system:

human skin model

Remarks:

model of human-derived epidermal keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: not applicable

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 (EPI-200) supplied by MatTek Corporation, Ashland MA, U.S.A
- Tissue batch number(s): 23280 kits X and J
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Date of certificate of analysis: 2016-01-13
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

TEMPERATURE USED FOR TEST SYSTEM
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 65 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both were supplied by MatTek Corporation.
- The DMEM medium was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Test for the interference with the MTT endpoint: The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 25 mg of the test item was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change was observed.
- Test for colour interference by the test material: The test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured substances in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, approximately 25 mg of the test item or 50 µl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (OD (540-570 nm)<1.0-3.0>): 1.825 +/- 0.105
- Barrier function (ET-50 <4.77-8.72 hrs>): 8.69 hrs
- Sterility: Sterile

PREDICTION MODEL / DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item 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 item is decreased below 15%.

A test item 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:

TEST MATERIAL
- Amount applied: 27.25 to 40.01 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

4 replicates per test item, negative control and positive control: 2 for the 3-minute exposure and 2 for the 1-hour exposure

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: individual values: 102 and 106%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: individual values: 96 and 103%

Results 3

Irritation / corrosion parameter:

other: Optical density

Run / experiment:

3-minute application

Value:

1.671

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: ± 0.043, individual values: 1.640 and 1.702

Results 4

Irritation / corrosion parameter:

other: optical density

Run / experiment:

1-hour application

Value:

1.883

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: ± 0.085, individual values: 1.823 and 1.944

Results 5

Irritation / corrosion parameter:

other: Maximum inter-tissue variability in viability between two tissues treated identically

Run / experiment:

3-minute application

Value:

3.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation / corrosion parameter:

other: Maximum inter-tissue variability in viability between two tissues treated identically

Run / experiment:

1-hour application

Value:

6.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

3-minute application:
- viability (percentage of control) (range):
negative control: 100 (101 and 99)
positive control: 15 (12 and 18)
- mean optical density:
negative control: 1.609 ± 0.022
positive control: 0.242 ± 0.066
- maximum inter-tissue variability in viability between two tissues treated identically
negative control: 1.9
positive control: 32.5
- maximum difference in percentage between the mean viability of two tissues and one of the two tissues
negative control: 0.9 and 1.0
positive control: 19.4 and 16.2
test material: 1.8 and 1.8


1-hour application:
- viability (percentage of control) (range):
negative control: 100 (96 and 104)
positive control: 13 (9 and 18)
- mean optical density:
negative control: 1.895 ± 0.094
positive control: 0.254 ± 0.121 (One of the tissues from another project was included (study plan deviation 1))
- maximum inter-tissue variability in viability between two tissues treated identically
negative control: 6.8
positive control: 50.3
- maximum difference in percentage between the mean viability of two tissues and one of the two tissues
negative control: 3.5 and 3.4
positive control: 33.6 and 25.1
test material: 3.2 and 3.1

Results:
The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.
The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 104% (102 and 106%) and 99% (96 and 103%) respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 15% (12 and 18%) and 13% (9 and 18%) after 1 hour exposure. The maximum inter-tissue variability in viability between two tissues treated identically was less than 7% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 4% for the negative control and test item. For the positive control, the maximum inter-tissue variability in viability between two tissues treated identically was less than 51% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 34%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test is valid and the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.