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-, ethyl ester, (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:

From 2012-02-28 to 2012-03-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

no

Remarks:

The study followed Good Laboratory Practice principles, however no study specific Quality Assurance procedures were performed and the report may not contain all of the elements required by GLP

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (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-carboxylate

EC Number:

618-843-3

Cas Number:

923604-57-3

Molecular formula:

C37H46N4O6S

IUPAC Name:

ethyl (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-carboxylate

Test material form:

solid

Details on test material:

- Name of test material (as cited in study reports): JNJ-38970191-AAA (T003009)
- Physical state: solid
- Appearance white solid

Specific details on test material used for the study:

- Batch n°: RT003009PFA061
- Analytical purity: 96.3%
- Expiration date: 22 November 2012
- Storage condition: at room temperature (ca. 20°C), dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Skin Irritation Test 42 hours using human epidermis skin constructs supplied by SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 12-EKIN-009
- The model 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 was 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.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The MTT reducing capability of the test substance, JNJ-38970191-AAA, was investigated by mixing 10 ± 2 mg of the test substance with 2 mL of 0.3 mg/mL MTT solution in duplicate.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.
- A control of 10 μL of distilled water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.
- 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.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No data

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1

DECISION CRITERIA
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).
If the mean tissue viability was greater than 50% of the negative control value, the sample was classed as non-irritant (EU classification) or No category (GHS classification)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg
A weight of 10 ± 2 mg 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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL


POSITIVE CONTROL
- Amount(s) applied (volume or weight):10 µL

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

After exposure to the test substance, 42 ± 1 hour incubation in maintenance medium at 37 ± 2£°C in a humidified atmosphere of 5% CO2 in air
Reduction of MTT: 3 hours
Extraction of tissues: 70 hours at 2-8°C protected from light

Number of replicates:

triplicates for test item, negative control and positive control each

Test system

Details on study design:

.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

100.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

SD of 3.5

Other effects / acceptance of results:

Negative control: tissue viability (mean ± SD): 100.0 ± 2.1 %
Positive control: tissue viability (mean ± SD): 15.3 ± 1.1 % -> irritant

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.

Any other information on results incl. tables

Possible reduction of MTT by test substance:

There was no change in the test substance, JNJ-38970191-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-38970191-AAA/water solution and water control were colourless after the 15 minute shaking period. The test substance, JNJ-38970191-AAA, had not shown any potential for colouring water.

Test substance pH:

The test substance, JNJ-38970191-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:

It was concluded that the test item, JNJ-38970191-AAA, with a mean tissue viability of 100.9 ± 3.5%, was predicted as non-irritant to the skin.