Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October 013 - 29 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 429. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material : [1-(3-Methoxy-phenyl)-ethyl]-methyl-amine Hydrobromide
- Physical state: white powder
- Analytical purity: 100.2 % (potentiometric assay)
- Lot/batch No.: I13025A
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 10 weeks old
- Weight at study initiation: 19.5-21.2 g
- Housing: Group caging / mice were provided with glass tunnel-tubes. Cage type: Type II. polypropylene / polycarbonate.
- Diet (e.g. ad libitum): ad libitum, ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25 and 50 % (w/v).
No. of animals per dose:
4 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200 (1% Pluronic) were tested. The maximum achievable concentration of test substance was 50 % (w/v) in DMF.
- Preliminary Irritation/Toxicity Test:
2 animals per dose were exposed to test item concentrations of 50 and 25 % (w/v) in DMF. The experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. No mortality or signs of systemic toxicity were observed. Test item precipitate was observed on the ears of the animals in the 50 % (w/v). No marked body weight loss was detected on the experimental animals. The revealing ear punch weights were within the historical control range. There were no indications of any irritancy at the site of application. The draining auricular lymph nodes of the animals were considered to be normal in both dose groups (visual observation). Based on these results, the 50 % (w/v) was selected as top dose for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
*That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in
control mice, as indicated by the stimulation index.
*The data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On day 6, each mouse received an injection of 250µl of sterile PBS (phosphate buffered saline) containing aprox. 20 µCi of 3HTdR. Five hours later, the mice were euthanized and the auricular lymph nodes were extracted from the animals. A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and the samples were prepared to be examined in a β-scintillation counter.

OBSERVATIONS:
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection).

EVALUATION OF RESULTS:
Radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes and corrected with the background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes). Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated.
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 stimulation index values were 2.49, 2.26 and 2.98 at concentrations of 50, 25 and 10 % (w/v), respectively. The negative control group DPN value was relatively low, hence the calculated SI values of the test item treated dose groups are slightly high but still below the biologically relevant threshold value.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Test item 50 % (w/v) in DMF: 1748.0 DPM Test item 25 % (w/v) in DMF: 1584.0 DPM Test item 10 % (w/v) in DMF: 2090.0 DPM

Any other information on results incl. tables

Clinical observation:

No mortality or signs of systemic toxicity were observed during the study. Test item precipitate was observed on the ears of the animals in the 50 % (w/v) dose group on Days 1-4, in the 25 % (w/v) dose group on Days 1-3 and in the 10 % (w/v) dose group on Days 2-3. There were no indications of any irritancy at the site of application.

Body weight:

No treatment related effects were observed on body weights.

Proliferation assay:

Test Group Name

Measured DPM / group

DPM

Number
lymph nodes

DPN

Stimulation Index

Background (5 % (w/v) TCA)

34

-

-

-

-

(-) control (DMF)

735

701.0

8

87.6

1.00

Test item 50 % (w/v) in DMF

1782

1748.0

8

218.5

2.49

Test item 25 % (w/v) in DMF

1618

1584.0

8

198.0

2.26

Test item 10 % (w/v) in DMF

2124

2090.0

8

261.3

2.98

(+) control (25 % (w/v) HCA

29017

28983.0

8

3622.9

41.35

The appearance of the lymph nodes was normal in the negative (vehicle) control group and in all test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

The DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range.

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 41.35) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance does not have sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42 (GLP study). Based on the results of the Preliminary Compatibility Test, the test item was formulated in N,N-dimethylformamide (DMF) at a highest achievable concentration of 50 % (w/v). The Preliminary Irritation / Toxicity Test was performed in CBA/J Rj mice using two doses: 50 and 25 % (w/v) in DMF. The 50 % (w/v) was selected as top dose for the main test. Based on these results, In the main twenty female CBA/J Rj mice were allocated to five groups of four animals each in the main test. Three groups received the test item at 50, 25 and 10 % (w/v) concentrations, the negative control group received the vehicle (DMF) and the positive control group received 25 % (w/v) HCA (dissolved in DMF). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or systemic clinical signs were observed during the study. Test item precipitate was observed on the ears of the animals in the 50 % (w/v) dose group on Days 1-4, in the 25 % (w/v) dose group on Days 1-3 and in the 10 % (w/v) dose group on Days 2-3. There were no indications of any irritancy at the site of application. The stimulation index values were 2.49, 2.26 and 2.98 at concentrations of 50, 25 and 10 % (w/v), respectively. All validity criteria were fulfilled. In conclusion, the test item was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.