METHYL (1R,2R,4S)-2-[HEX-5-EN-1-YL(METHYL)CARBAMOYL]-4-({7-METHOXY-8-METHYL-2-[4-(PROPAN-2-YL)-1,3-THIAZOL-2-YL]QUINOLIN-4-YL}OXY)CYCLOPENTANECARBOXYLATE

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-01-18 to 2016-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well documented GLP study performed according to OECD guideline 439 and EU method B.46. No deviations were recorded.

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

methyl (1R,2R,4S)-2-[(hex-5-en-1-yl)(methyl)carbamoyl]-4-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)cyclopentane-1-carboxylate

EC Number:

922-147-8

Cas Number:

1042695-87-3

Molecular formula:

C32H41N3O5S

IUPAC Name:

methyl (1R,2R,4S)-2-[(hex-5-en-1-yl)(methyl)carbamoyl]-4-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)cyclopentane-1-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-39125593-AAA (T003019)
- Physical state: solid
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15FB2925
- Expiration date of the lot/batch: 2017-05-31 (retest date)
- Date of manufacture: 2016-06-01
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under test conditions: Not available
- Solubility and stability of the test substance in the solvent/vehicle: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the solid test item was applied directly on top of the skin tissue.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Source: EPISKIN Small Model(TM) (EPISKIN-SM(TM), 0.38 cm², Lot no.: 16-EKIN-003), SkinEthic Laboratories, Lyon, France.
- This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

ENVIRONMENTAL CONDITIONS
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 66 - 87%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 36.7°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.

TEST FOR THE INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
- A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
- The test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, at least 10 mg of the test item was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
- 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, at least 10 mg of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

APPLICATION/TREATMENT OF THE TEST ITEM
The skin was moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (30.0 to 40.1 mg; with a glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µl PBS (negative control) and 3 tissues with 25 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

INTERPRETATION
- A test item is considered irritant in the skin irritation test (category 2) if: the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test (no category) if: the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ELECTRONIC DATA CAPTURE
- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA) for temperature and humidity.
- Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM: 30.0 to 40.1 mg

NEGATIVE CONTROL (PBS): 25 µL

POSITIVE CONTROL (5% SDS): 25 µL in PBS

Duration of treatment / exposure:

15 +/- 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 tissues per test item together with negative and positive controls

Test animals

Species:

rabbit

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

114

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

range: 101 - 121 %

Results 2

Irritation / corrosion parameter:

other: Optical density

Run / experiment:

1

Value:

1.024

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

range: 0.907 - 1.086

Other effects / acceptance of results:

mean tissue viability (percentage of control):
negative control: 100 +/- 9.1%
positive control: 17 +/- 0.8 %

mean optical density:
negative control: 0.898 +/- 0.082
positive control: 0.154 +/-0.007

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 no colour changes were observed it was concluded that the test item did not interact with the MTT endpoint.

Interpretation:
All viability of all replicates was within one category.

Results:
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 114% (101 to 121%). Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 17% (16 to 18%). The absolute mean OD570 of the negative control tissues was within 0.6 and 1.5. The standard deviation values of the percentage viability of three tissues treated identically were 9.1%, 0.8% and 11.3% for the negative, positive control and the test item respectively, indicating 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 non-irritant in the in vitro skin irritation test under the experimental conditions described in the report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.