Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well perfomed OECD study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
purity: 94.7 %

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.5 - 22.7 g
- Housing: single cages
- Diet: pelleted standard diet, (Harlan Laboratories GmbH, D-33178 Borchen) ad libitum
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: At least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-3
- Humidity (%): 30 -70
- Air changes (per hr):
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
other: ethanol:deionised water (70 : 30)
Concentration:
0, 1, 5, 10 %
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
The highest test item concentration, which can be technically used was a 10 % suspension in methyl ethyl ketone. Due to the possibility of a reaction of the test item with the vehicle, ethanol:deionised water (70+30) was choosen as vehicle by the sponsor. A suspension of 10% could be achieved after warming for 30 min. at 37°C.
Two mice were treated with concentrations of 5, and 10 % on both ears each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application and on day corresponding to the day of preparation of the main experiment. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1, 5 and 10 % (w/v) in ethanol:deionised water (70+30). The application volume, 25 µl, was spread over the entire dorsal surface (diameter about 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle
alone (control animals).
Five days after the first topical application, all mice were administered with 250 µl of 85.2 µCi/ml 3HTdR (corresponds to 21.3µCi 3HTdR per mouse) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred
to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid.
The level of 3HTdR incorporation was then measured on a ß-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).


- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Positive control substance: alpha-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1);
concentrations 5, 10, 25 %
Experiment performed in July 2008.
The EC3 value was estimated to be < 5%. A precise calculation of the EC3 value was not possible since the S.I. was above the threshold of 3 even at the lowest tested concentration. The obtained values are within the expected range and the positive control experiment is considered fully valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Test item Calculation concentration Group Measurement number of DPM per % (w/v) DPM DPM-BG lymph nodes lymph node S.I. -- BG I 21 -- -- -- -- -- BG II 19 -- -- -- -- 0 1 4058 4038 8 504.8 1 2 3619 3599 8 449.9 0.89 5 3 3875 3855 8 481.9 0.95 10 4 2920 2900 8 362.5 0.72 BG = Background (1 ml 5% trichloroacetic acid) in duplicate 1 = Control Group 2-4 = Test Group S.I. = Stimulation Index The EC3 value could not be calculated, since all S.I.´s are below 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see below

Any other information on results incl. tables

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

The measured ear weights of all animals treated were recorded after sacrifice. A relevant increase in ear weights was not observed.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item Aminoanisoylaminobenzamid Hydrochlorid trocken was not a skin sensitiser under the described conditions.
Executive summary:

In the study the test item Aminoanisoylaminobenzamid Hydrochlorid trocken dissolved in ethanol:deionised water (70+30) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay (OECD 429)was performed using test item concentrations of 1, 5 and 10 %. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 0.89, 0.95 and 0.72 were determined with the test item at concentrations of 1, 5 and 10 % in ethanol:deionised water (70+30), respectively.

The test item Aminoanisoylaminobenzamid Hydrochlorid trocken was not a skin sensitiser in this assay.