Cyclopenta[c]cyclopropa[g][1,6]diazacyclotetradecine-12a(1H)-carboxylic acid, 2,3,3a,4,5,6,7,8,9,11a,12,13,14,14a-tetradecahydro-2-[[7-methoxy-8-methyl-2-[4-(1-methylethyl)-2-thiazolyl]-4-quinolinyl]oxy]-5-methyl-4,14-dioxo-, (2R,3aR,10Z,11aS,12aR,14aR)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-02-28 to 2012-03-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study performed in accordance with the OECD Guideline 439 and the Standard Operating Procedure, In Vitro Skin Irritation Test: Human Epidermis Model (L’Oreal 2009).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

other: Standard Operating Procedure, In Vitro Skin Irritation Test: Human Epidermis Model

Deviations:

not specified

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1R,4R,6S,7Z,15R,17R)-17-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)-13-methyl-2,14-dioxo-3,13-diazatricyclo[13.3.0.0^{4,6}]octadec-7-ene-4-carboxylic acid

EC Number:

618-844-9

Cas Number:

923604-58-4

Molecular formula:

C35H42N4O6S

IUPAC Name:

(1R,4R,6S,7Z,15R,17R)-17-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)-13-methyl-2,14-dioxo-3,13-diazatricyclo[13.3.0.0^{4,6}]octadec-7-ene-4-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-38940642-AAA (T003010)
- 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: RT003010PFA121
- Expiration date of the lot/batch: 2012-12-01
- Purity: 96.0%
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ca. 20°C), dark
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: none

FORM AS APPLIED IN THE TEST: white powder

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

Vehicle:

unchanged (no vehicle)

Details on test system:

The EPISKIN Skin Irritation Test-42 hours using human epidermis skin constructs was supplied by SkinEthic Laboratories, Lyon, France, and consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38 cm². The EPISKIN kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar. On receipt, the kit contents were checked and the inserts with tissues on agarose were stored at room temperature until use. The kit was used within the expiry date indicated by the supplier (expiry date: 5 March 2012). The maintenance medium was pre-warmed to 37º C. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.



RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 99-KERA-037, 00-KERA-014, 02-KERAMR-005, 09-KERA-003
- It consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38 cm².
- The EPISKIN kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar. On receipt, the kit contents were checked and the inserts with tissues on agarose were stored at room temperature until use. The kit was used within the expiry date indicated by the supplier (expiry date: 5 March 2012). The maintenance medium was pre-warmed to 37º C. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

REDUCTION OF MTT BY TEST SUBSTANCE:
It is possible that a test substance may reduce MTT, resulting in the production of a blue colour without any involvement of cellular mitochondrial dehydrogenase. Since the test substance is rinsed off the tissue before the MTT assay, this is usually avoided. However, it is possible that small amounts of test substance may be present after washing or be released through the tissue into the MTT medium. If the mixture with the test substance turns blue/purple after approximately 3 hours incubation at 37 ± 2ºC in 5% CO2 in air, MTT reduction may have occurred.
The MTT reducing capability of the test substance, JNJ-38940642-AAA, was investigated by mixing 10 ± 2 mg of the test substance with 2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 μL of distilled water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.

CHECK FOR COLOURING POTENTIAL OF TEST SUBSTANCE:
The test substance, JNJ-38940642-AAA, was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). Evaluation was achieved by mixing 0.112 g of the test substance, JNJ-38940642-AAA, with 101 μL of distilled water in a transparent container to give 10 mg/90 μL water. 100 μL of distilled water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the colour of the solution was assessed by eye.

PREPARATION/APPLICATION OF SAMPLES:
After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 ± 0.5 minutes with the test substance, negative or positive control at room temperature. The positive and negative controls were in liquid form and were applied by dispensing a volume of 10 μL over each tissue using a positive displacement pipette. A weight of 10 ± 2 mg of the test item was dispensed over each tissue using glass weighing boats. The tissues were wetted with 5 μL of distilled water prior to application of the test substance. The test substance was spread over the surface of the tissue using a curved spatula.

TEST PROCEDURE:
A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 7 minutes application time.
After 15 ± 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
At the end of 3 hours ± 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube.
When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration). The tissues were extracted by storing at 2-8 ºC, protected from light, for 70 hours. After formazan extraction, duplicate 200 μL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

DATA INTERPRETATION:
If the mean tissue viability was equal to or less than 50% of the negative control value, the sample was classed as Irritant R38 (EU classification) or Category 2 (GHS classification).

COMPUTER SYSTEMS:
The computer system used in this study was as follows: Pristima – Test substance management

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10±2 mg

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 ± 1 hours

Number of replicates:

3 tissues per test item together with negative and positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item after 15 minutes of treatment

Value:

98.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

SD = 7.9 %

Other effects / acceptance of results:

NEGATIVE AND POSITIVE CONTROLS
The mean absorbance of the triplicate negative control values was 0.873 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 2.1 which was below the maximum value of 18.
The percentage mean viability of the positive control was 15.3 ± 1.1 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

POSSIBLE REDUCTION OF MTT BY TEST SUBSTANCE
There was no change in the test substance, JNJ-38940642-AAA/MTT solution or the water control/MTT solution after three hours incubation in the dark at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The test substance had not interacted with the MTT.

CHECK FOR COLOURING POTENTIAL OF TEST SUBSTANCE
The test substance, JNJ-38940642-AAA/water solution and water control were colourless after the 15 minute shaking period. The test substance had not shown any potential for colouring water.

TEST SUBSTANCE pH
The test substance, JNJ-38940642-AAA was diluted to 10% w/v with distilled water to obtain an aqueous solution for pH measurement. The pH of the test substance, measured using pH indicator paper, was approximately 7.0.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the in vitro study, T003010 is observed to be non-irritating to the skin.