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 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:

other: Well documented GLP-study performed accroding to the OECD guideline n°429 and EU Method B.42.

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:

(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

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 Rives UK Ltd.
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 17.9 to 21.9 g
- 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): ad libitum, potable water from public supply via polycarbonate bottles fitted with sipper tubes
- Acclimation period: 6 days prior the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 °C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2011-10-06 To: 2011-10-31

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10 and 25% w/v (preliminary investigations)
2.5, 5 or 10% w/v (main study)

No. of animals per dose:

2 female mice/dose level (Preliminary investigations)
4 female mice/dose level and per control (Main study)

Details on study design:

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

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 β-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)

Statistics:

no data

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.8

Test group / Remarks:

2.5% w/v group

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

5% w/v group

Results 3

Parameter:

SI

Value:

0.9

Test group / Remarks:

10% w/v group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
2.5% -7084.70 dpm 5 % - 8793.50 dpm 10 % w/v -7728.80 dpm The dpm/node (test/control ratios) obtained for 2.5, 5 and 10 % w/v were 885.59, 1099.19 and 966.10 dpm/node respectively.

DETAILS ON STIMULATION INDEX CALCULATION
see results table

EC3 CALCULATION
The EC3 value was reported to be greater than the highest concentration tested (10% w/v).

CLINICAL OBSERVATIONS:
Mortality and Clinical Signs
Preliminary investigations : There were no deaths and no signs of ill health or toxicity observed during this study.
At 10% w/v, wet fur on the head was noted following each dosing occasion. In addition, white dose residue on the head was seen post dose on Days 2 and 3 and it was also present on Day 4.
At 25% w/v, wet fur on the head and white dose residue on the head was noted following each dosing occasion. In addition, white dose residue on the head was also present on Days 4 and 5.
Main phase: There were no deaths and no signs of ill health or toxicity observed during this study.
Wet/Greasy fur on the head was noted in all the vehicle control animals, treated groups and positive control animals following each dosing occasion, 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 was seen post-dose on Days 2 and 3 in all females in Group 6 (10% w/v) and on Day 4 in two females in Group 6.

Dermal reactions
Preliminary investigations: No dermal irritation was observed on the ears of any mouse at each dose level on Days 1 to 6.
Main phase: No signs of dermal irritation were seen on the ear during the study.

Measurement of ear thickness
Preliminary investigations: There was evidence of an effect of treatment on ear thickness at 25% w/v (% difference from Day 1 went up to 46%). There was no evidence of an effect of treatment on ear thickness at 10% w/v.

BODY WEIGHTS
Preliminary investigations: There was no indication of an overt effect of treatment on bodyweight gain. On the basis of the results from the preliminary investigation, 10% w/v was considered a suitable high concentration for administration in the main phase of the study.
Main phase: A loss in bodyweight was noted for one mouse (No. B8) and no bodyweight gain was seen in one further mouse (No. B23) over the study period, however, a small loss in bodyweight or no weight gain 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:

JNJ-38940642-AAA is not regarded as a potential skin sensitizer. Thefore the test substance is not to be classified