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 sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-06 to 2011-11-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

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
- Batch number: RT003019PFP201
- Expiration date of the lot/batch: 06 July 2011 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (15-25°C) protected from light
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was used as supplied. A conversion factor of 1.02 was applied and all formulations were prepared on the day of dosing at the required concentration(s).
A vehicle trial was conducted and JNJ-39125593-AAA showed the following:
At 100% w/v, the material was too bulky to achieve a formulation in any vehicle tested and therefore this concentration was not suitable for use on this study.
At 50% w/v the formulations in all vehicles were thick pastes or too bulky to formulate and not suitable for dosing.
At 25% w/v, the formulation in acetone:olive oil (4:1 v/v) was an off white suspension, the formulation in dimethylformamide was a very fine pale brown suspension, the formulation in methyl ethyl ketone was a clear pale
brown solution, the formulation in propylene glycol was a very thick off white suspension and the formulation in dimethylsulphoxide gave a thick off white suspension. With the exception of the formulation in propylene
glycol, all of these formulations were suitable for dosing.
At 10% w/v, the formulation in acetone:olive oil (4:1 v/v) was a clear yellow solution, the formulation in dimethylformamide was a clear pale brown solution, the formulation in propylene glycol was a thick of white, bitty
suspension and the formulation in dimethylsulphoxide was an off white suspension. All of these formulations were suitable for dosing. Based on the results at 25% w/v, methyl ethyl ketone was chosen as the vehicle for this study.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: 24 healthy female CBA/Ca mice; Charles River UK Ltd
- Age prior to dosing on day 1: 8-12 weeks
- Weight prior to dosing on day 1: 18-21.8 grams
- Housing: inside a barriered rodent facility, designed and operated ro minimise the entry of external biologial and chemical agents and to minimise the transference of such agents between rooms; pair housed in solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved wood flake bedding, additionally Nestlest and a plastic shelter were included for environmental enrichment.
- Diet (e.g. ad libitum): the animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants
- Water (e.g. ad libitum): potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. The results of these various analyses did not provide evidence of contamination.
- Acclimation period: at least 6 days prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%): 40-70%
- Air changes (per hr): the animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to the atmosphere and re-circulated.
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Remarks:

25% w/v

Concentration:

0, 5, 10 and 25% (w/v)

No. of animals per dose:

Preliminary investigation: 2 females/group
Main study: 4 females/group; 3 test groups and 1 control group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: no data
- Irritation: No dermal irritation was observed on the ears of any mouse on Days 1 to 6.
- Systemic toxicity: There were no deaths and no signs of ill health or toxicity observed during this study.
At 10% w/v, greasy fur on the head was noted following each dosing occasion. In addition, white dose residue on the ears was seen post dose from Day 2 and on Days 4 and 5. All signs had recovered by Day 6.
At 25% w/v, greasy fur on the head was noted following each dosing occasion. In addition, white dose residue on the ears was seen post dose from Day 1, prior to dosing from Day 2 and on Days 4 to 6.
- Ear thickness measurements: There was no evidence of an effect of treatment on ear thickness.
- Erythema scores: 0 in all animals for day 1 to 6

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Groups of four female mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µl of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (days 1-3). The test substance was applied using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette. Further groups of four mice received the vehicle alone or the positive control substance in the same manner.
- Criteria used to consider a positive response: results for each treatment group were expressed as the Stimulation Index (S.I.). This was derived by dividing the mean DPM/mouse for each treated group or the positive control group by the mean DPM/mouse in the vehicle control group. If the SI is 3 or more, the test substance is regarded as a skin sensitizer. The positive control group is expected to give an SI of 3 or more to demonstrate the validity of the study.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: If the SI is 3 or more, the test substance is regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance, was prepared for administration as a series of graded concentrations in the vehicle, by direct dilution. The test substance was used as supplied. A conversion factor of 1.02 was
applied and all formulations were prepared on the day of dosing at the required concentration(s). The absorption of the test substance was not determined. Determination of the homogeneity, stability and purity of the test substance or test substance formulations were not undertaken as part of this study.
The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette. Further groups of four mice received the vehicle alone or the positive control substance in the same manner.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

No statistical analysis performed.

Results and discussion

Positive control results:

The SI for the positive control substance hexyl cinnamic aldehyde (HCA), was 12.5 which demonstrates the validity of this study.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.4

Test group / Remarks:

5% w/v group

Results 2

Parameter:

SI

Value:

1.7

Test group / Remarks:

10% w/v group

Results 3

Parameter:

SI

Value:

2.8

Test group / Remarks:

25% w/v group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
5% w/v group: DPM/node: 730.13 (8 lymph nodes)
10% w/v group: DPM/node: 865.88 (8 lymph nodes)
25% w/v group: DPM/node: 1429.06 (8 lymph nodes)

DETAILS ON STIMULATION INDEX CALCULATION
The SI (test/control ratios) obtained for 5, 10 and 25% w/v were 1.4, 1.7 and 2.8 respectively. As a SI of 3 or more was not recorded for any of the concentrations tested, is not considered to have the potential to cause skin sensitization.

EC3 CALCULATION
Based on the results of this study the EC3 value was reported to be greater than the highest concentration tested (25% w/v).

MORTALITY AND CLINICAL OBSERVATIONS:
There were no deaths and no signs of ill health or toxicity observed during this study.
Greasy fur on the head was noted for all animals following each dosing occasion and also prior to dosing on Days 2 and 3 and on Day 4 for Group 7 (positive control), this was related to unoccluded dermal administration of a
liquid formulation/vehicle and not an effect of the test substance. In addition, white dose residue on the ears was seen post-dose on Days 2 and 3 in Groups 5 and 6 (10 and 25% w/v) and post-dose on Day 1, prior to dosing
on Day 3 and on Days 4, 5 and 6 for Group 6.

DERMAL REACTIONS
No signs of dermal irritation were seen on the ear during the study.

BODY WEIGHTS
There was no indication of an effect of treatment on bodyweight gain. A loss in bodyweight was noted for two mice (Nos. D18 and D24) over the study period, however, a small loss in bodyweight is not uncommon in young laboratory mice and is not considered to be an effect of treatment.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was not regarded as a potential skin sensitizer.