Registration Dossier

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 423) and in compliance with GLP.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
Identification: IN 00078280
Supplier: Boehringer Ingelheim Pharmaceuticals
Batch No.: 7889-142
Expiration Date: Dec 2007
Physical Description: Off white powder
Composition/Purity: 100% (NMR)
Stability: Stability data on bulk test material was not
provided by the Sponsor.
Storage Conditions: Room temperature

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Jackson Laboratories, Bar Harbor, ME 04609
- Age at study initiation: 8-9 weeks at start of dosing; records of dates of birth of animals used in this study are retained in the Calvert archives
- Weight at study initiation: 18 to 25 grams at the outset (Day 1) of the study
- Housing: Animals were group housed (5 per cage) upon receipt in compliance with National Research Council “Guide for the Care and Use of Laboratory Animals”. The room in which the animals were kept was documented in the study records. No other species were kept in the same room.
- Diet (e.g. ad libitum): Animals had access to Certified Rodent Diet 7012C ad libitum. The lot number(s) and specifications of each lot used are archived at Calvert.
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Study animals were acclimated to their housing for a minimum of 6 days prior to their first day of dosing.

- Temperature (°C): 20 to 30.6 °C
- Humidity (%): 30 to 64%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
10 %, 25 %, 50 %,
No. of animals per dose:
25 total; 5 per concentration; 5 per positive and negative control, respectively
Details on study design:
- Compound solubility: The vehicle and dose levels were selected by conducting a solubility test. In order of preference the vehicles of choice were acetone/olive oil (4:1 v/v) (AOO), dimethylformamide (DMF), methyl ethyl ketone (MEK) propylene glycol (PG), dimethyl sulfoxide (DMSO). The recommended doses were
selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% (w/v) etc, the top dose being the highest level that could be achieved.
- Irritation: The doses for this assay were selected from the concentrations series 100%, 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were chosen, the top one being the highest level that could be achieved based on solubility while avoiding systemic toxicity and excessive local irritation.

- Name of test method: LLNA
- Criteria used to consider a positive response: Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control as indicated by the SI.

TREATMENT PREPARATION AND ADMINISTRATION: Groups of 5 CBA/J female mice were treated on the dorsal surface of both ears once per day for 3 days with IN 00078280 at 10, 25 or 50%, with the vehicle (acetone/olive oil (4:1, v/v) [AOO]), or with the positive control (35% Hexylcinnamaldehyde [HCA]). On Day 6, the mice were injected, i.v., with 20 μCi of 3H-thymidine in sterile saline. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were precipitated with 5% trichloroacetic acid (TCA) and the pellets counted in a ß-scintillation counter to determine incorporation of the 3Hthymidine. This study was run three times since in the first two runs the vehicle control results were high resulting in SI values <3 for the positive control group, which invalidated the data.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The mean DPM for each group was evaluated using SYSTAT version 9.01, developed by SPSS, Inc. Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI).
Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM and body weight data were made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett’s test was used to determine the degree of significance from the control means.

Results and discussion

Any other information on results incl. tables

 Group  Treatment  Dose  DPM (mean +- sem)  SI  Result1
 1  AOO  -  1166 +-396  -  negative
 2  IN00078280  10%  472 +-65  0.4  -
 3  IN00078280  25%  1391 +-242  1.2  -
 4  IN00078280  50%  646 +-232  0.6  -
 5  HCA  35%  4600 +-671***  3.9  +

1Test/control ratio of 3.0 or greater represents a positive result

***Statistically significant difference when log DPM compared to the vehicle control group (group 1) (p < 0.001)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU
A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control group. Thus, a stimulation index (SI) ≥ 3.0 is regarded as a positive response. Although the dosing solution concentrations were not verified, treatment with IN 00078280 at nominal concentrations of 10, 25 or 50% did not result in an SI of 3 or greater.
Therefore, based on the data from this study, IN 00078280 is not considered to have skin sensitizing potential.