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 sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2011-10-06 to 2011-11-02

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:

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:

SOURCE OF TEST MATERIAL
- Source: delivered by the sponsor (Tibotec Pharmaceuticals Ltd., a member of the J&J group of companies, Cork, Ireland)
- Lot/batch No.of test material: RT003009PFA061
- Expiration date of the lot/batch: 22 November 2012 (retest date)
- Purity: 96.3% w/w

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: Determination of the homogeneity, stability and purity of the test substance or test substance formulations were not undertaken as part of this study.
- Solubility and stability of the test substance in the solvent/vehicle:
At 2.5, 5 or 10% w/v, the formulations in acetone:olive oil (4:1 v/v), dimethylformamide, methyl ethyl ketone and propylene glycol gave an offwhite suspension and the formulation in dimethylsulphoxide gave an off white/cream suspension. All of these formulations were suitable for dosing. Based on the results at 25% w/v, acetone:olive oil (4:1 v/v) was chosen as the vehicle for this study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test substance, JNJ-38970191-AAA, 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 1.04 was applied and all formulations were prepared on the day of dosing at the required concentration(s).
FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 17.6 - 20.5g
- Housing: Inside a barriered rodent facility, designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. The animals were randomized within the treatment groups. They were pair housed, in solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved wood flake bedding, additionally Nestlets and a plastic shelter were included for environmental enrichment.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: at least 6 days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily and records are archived with the departmental raw data.
Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary investigations: 10 and 25% w/v in vehicle
Main study: 5, 10 and 25% w/v in vehicle

No. of animals per dose:

Preliminary investigations: 2 female mice per dose
Main study: 4 female mice per dose and per control

Details on study design:

RANGE FINDING TESTS:
Two female mice (per concentration) received a daily application of 25 µL of 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.

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.

Administration of 3H-methyl Thymidine:
In the main phase of the study, five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline containing 3H-methyl thymidinea (3HTdR: 80 µCi/mL) giving a nominal 20 µCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.

TERMINAL STUDIES
Termination:
The mice in the preliminary investigation were humanely killed by carbon dioxide asphyxiation on Day 6 of the study. The carcasses were discarded and no further investigations were carried out with these animals. In the main phase of the study, five hours following the administration of 3HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised and pooled for each experimental group. 1.0 mL of phosphate buffered saline was added to the pooled lymph nodes for each group. The animals were then discarded and no further investigations were carried out. The following procedures were carried out with the lymph nodes from these animals only.

Preparation of Single Cell Suspensions:
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The pooled LNCs were then washed by adding 10 mL phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash.

Determination of Incorporated 3H-methyl Thymidine:
After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by beta-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio).

Positive control substance(s):

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

Results and discussion

Positive control results:

The Stimulation Index for the positive control substance hexyl cinnamic aldehyde (HCA), was 10.2 which demonstrates the validity of this study.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.2

Test group / Remarks:

5% w/v

Results 2

Parameter:

SI

Value:

1.3

Test group / Remarks:

10 % w/v

Results 3

Parameter:

SI

Value:

1.2

Test group / Remarks:

25% w/v

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
5% w/v group: dpm: 8395.40 (8 lymph nodes)
10% w/v group: dpm: 5147.70 (8 lymph nodes)
25% w/v group: dpm: 4600.50 (8 lymph nodes)

DETAILS ON STIMULATION INDEX CALCULATION
The SI (test/control ratios) obtained for 5, 10 and 25% w/v of the test item were 2.2, 1.3 and 1.2 respectively. As a SI of 3 or more was not recorded for any of the concentrations tested, the test item 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).

CLINICAL OBSERVATIONS:
Preliminary investigations:
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 1. All signs had recovered by Day 4.
At 25% w/v, white dose residue on the ears was seen post-dose from Day 1. In addition, greasy fur on the head was noted following dosing on Day 3. All signs had recovered by Day 4.

Main phase:
There were no deaths and no signs of ill health or toxicity observed during this study.
Greasy fur on the head was noted following each dosing occasion for all animals in all groups and prior to dosing on Days 2 and 3 and on Day 4 in the majority or all animals in 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 from Day 1 for all females in the treated groups.

MEASUREMENT OF EAR THICKNESS:
Preliminary investigations:
There was no evidence of an effect of treatment on ear thickness.

DERMAL REACTIONS:
Preliminary investigations:
No dermal irritation was observed on the ears of any mouse on Days 1 to 6.

Main phase:
No signs of dermal irritation were seen on the ear during the study.

BODY WEIGHTS
Preliminary investigations:
No bodyweight gain was noted for one mouse (No. C2) over the study period, however, this is not uncommon in young laboratory mice and was not considered to be an effect of treatment.
On the basis of the results from the preliminary investigation, 25% w/v was considered a suitable high concentration for administration in the main phase of the study.

Main phase:
There was no indication of an effect of treatment on bodyweight gain.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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