Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2011-12-01 to 2011-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to the OECD Guideline for Testing of Chemicals No. 429 and the EEC Methods for the determination of toxicity and other health effects (No. 440/2008. Part B, Method B.42.).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
bis(ethyl (1R,2S)-1-amino-2-ethenylcyclopropane-1-carboxylate); sulfuric acid
EC Number:
689-958-4
Cas Number:
1173807-85-6
Molecular formula:
C8H13NO2.0.5H2SO4
IUPAC Name:
bis(ethyl (1R,2S)-1-amino-2-ethenylcyclopropane-1-carboxylate); sulfuric acid
Details on test material:
- Name of test material (as cited in study report): JNJ-31052047-ABI
- Physical state: solid
- Analytical purity: 100.1%
- Purity test date: 06 July 2011
- Lot/batch No.: 00549620
- Storage condition of test material: At room temperature (approximately 15-25°C)

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 to 12 weeks
- Weight at study initiation: 17.0 - 20.3 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
- Acclimation period: 7 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.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10 and 25% w/v in vehicle
No. of animals per dose:
Preliminary investigations: 2 mice per dose
Main study: 4 mice per dose and per control

Details on study design:
RANGE FINDING TESTS

Two female mice (per concentration) received a daily application of 25µL 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 thymidine (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

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The SI (test/control ratios) obtained for 5, 10 and 25% w/v JNJ-31052047-ABI were 1.9, 4.6 and 8.4 respectively. As a SI of 3 or more was recorded for two of the concentrations tested, JNJ-31052047-ABI was considered to have the potential to cause skin sensitization. Based on the results of this study the EC3 value is calculated to be 7% w/v. The SI for the positive control substance hexyl cinnamic aldehyde (HCA), was 3.9 which demonstrates the validity of this study.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The dpm obtained for 5, 10 and 25% w/v JNJ-31052047-ABI were 1277.35, 3132.85 and 5695.75 dpm respectively. The dpm/node (test/control ratios) obtained for 5, 10 and 25% w/v JNJ-31052047-ABI were 159.67, 391.67 and 711.97 dpm/node respectively.

Any other information on results incl. tables

Mortality and Clinical Signs:

Preliminary investigations: There were no deaths and no signs of ill health or toxicity observed during this study. At 10 and 25% w/v, greasy fur on the head was noted following each dosing occasion. All signs had recovered by Day 4. This sign was considered to be related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

Main study: 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, pre-dose on Days 2 and 3 for all animals in Group 7 and on Day 4 in all animals in Groups 6 and 7. In addition, greasy fur was also seen in 2 animals in Group 7 on Day 5 and one of these animals had hairloss on Day 6. Greasy fur was considered to be related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

Dermal reactions: No dermal irritation was observed on the ears of any mouse on Days 1 to 6, for both preliminary investigations and the main study.

Measurement of ear thickness: There was no evidence of an effect of treatment on ear thickness during the preliminary investigations.

Body Weights: There was no indication of an effect of treatment on bodyweight gain, for both preliminary investigations and the main study. 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.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
JNJ-31052047-ABI is regarded as a potential skin sensitizer. The EC3 value was calculated to be 7% w/v.