Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
use of potentially corrosive concentrations.
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
use of potentially corrosive concentrations.
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
EPA 712-C-03-197, March 2003
Deviations:
yes
Remarks:
use of potentially corrosive concentrations.
GLP compliance:
yes
Type of study:
Buehler test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-Ethylpiperidin
- Physical state: liquid, clear, colourless
- Analytical purity: 100%
- Lot/batch No.: 0102562158
- Expiration date of the lot/batch: January 17,2008
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Crl:HA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory Animal Breeders, Kisslegg, Germany
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 346 - 400 g
- Housing: two or five per cage kept in Noryl cages, Tecniplast Deutschland GmbH, Hohenpeissenberg, Germany
- Diet: "PROVIMI KLlBA 3420 - Maintenance Diet for Guinea Pigs" supplied by PROVOMI KLlBA AG, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 (possibly drifting higher at outdoor temperatures above 24°C)
- Humidity (%): 40-70
- Air changes (per hr): ≥ 10 times per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
1st induction: 6%; 2nd and 3rd induction: 3%; Challenge: 3%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
1st induction: 6%; 2nd and 3rd induction: 3%; Challenge: 3%
No. of animals per dose:
20 females (in test groups)
10 females (controls)
Details on study design:
RANGE FINDING TESTS:
In a first dose range-finding test three concentrations and the vehicle were tested in each case on five guinea pigs (animal nos. 1-5). The suitable areas of the body were shaved one day (24 hours) before the treatment. The patches loaded with 0.5 ml of the test item formulations (25% and 50%), the test item (100%) or the vehicle were applied to each animal under occlusive conditions for 6 hours. At the end of the exposure period the animals showed following clinical signs: temporary tremor, spasmodic state and prostration.
The treated skin areas appeared corroded. Because of the clinical signs and the strong skin effects the animals were sacrificed.

In a second dose range-finding test three additional concentrations and the vehicle were tested. Each concentration and the vehicle were tested on three guinea pigs. The right flanks of the body were shaved one day (24 hours) before the treatment. The patches loaded with 0.5 ml of the test item formulations (1%; 3% and 6%) or the vehicle were applied to each animal under occlusive conditions for 6 hours. At the end of the exposure period, the remaining test item was removed with sterile physiological saline solution. Twenty-one hours later the treated areas were shorn.
The dermal reactions were evaluated 30 and 54 hours after the start of the application.
Because of the toxic effect of the test item observed during the first dose range finding study, each animal was only treated with one test item concentration in this study.
There were no skin effects in the animals treated with 1, 3, and 6 % test item at the evaluation time points of 30 and 54 hours after start of application.

Because of this negative result a 12% test item formulation was tested in a third dose range-finding test. The left flanks of 4 animals of the second dose range-finding test were shaved one day (24 hours) before the treatment. The third dose range-finding test was carried out as described for the second study. At 30 and 54 hours readings, the treatment area was black coloured and appeared corroded.
Thereafter, one further animal of the second dose rangefinding test was shaved on the right and left flank and treated with three test item concentrations to check the overall toxicity of the test item at the concentrations selected for the main study.
With the 12 % test item formulation the treatment area was black coloured in places and appeared corroded, as observed in the third range finding study. The animal showed no clinical signs.

Selection of the dose for the inductions:
Based on the results of the dose range-finding studies, the following concentration was selected for the first induction: 6%
Because of the strong skin effects after the 1st induction the dose was reduced to 3% for the 2nd and 3rd induction.

The test item concentrations for the challenge was determined in a dose range-finding study using 2 guinea pigs which were treated during the inductions in the same manner as the control animals. The patches loaded with 0.5 ml of test item formulations or the vehicle were applied to each animal under occlusive conditions for 6 hours. At the end of the exposure period, the remaining test item was removed with sterile physiological saline solution. Twenty-one hours later the treated areas were shorn. The dermal reactions were evaluated 30 and 54 hours after the start of the application.
There were no skin effects in the animals at the evaluation time points of 30 and 54 hours after treatment with 1, 3, and 6 % test item.
Based on the results of the dose range-finding study, the concentration of 3 % was selected for the challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
The animals were dermally treated with the test item three times at intervals of seven days. The suitable areas of the body were shaved one day (24 hours) before each treatment. After removal of the patches, any remaining test item was removed with sterile physiological saline solution.
- No. of exposures: 3 times at intervals of seven days
- Exposure period: 6 h
- Test groups: Hypoallergic patches loaded with the test item in pysiol. saline were held in place on the skin using "ORABAND"® adhesive plaster
- Control group: Hypoallergic patches were loaded with physiol. saline
- Site: left flank (1st induction); right flank (2nd and 3rd induction)
- Frequency of applications: every 7 days
- Duration: 0 - 21 days
- Concentrations: 6 % (1st induction); 3 % (2nd and 3rd induction)
- Volume applied per animal: 0.5 mL.
The treatment areas were visually assessed 30 hours after initiation of exposure. For this, the treatment areas were not shorn or chemically depilated.

B. CHALLENGE EXPOSURE
The challenge was performed two weeks after the last dermal induction.
For the challenge backs and right flanks of the animals were shorn 30 minutes prior to the challenge treatment.
The volume applied per animal was 0.5 mL.
At the end of the exposure period, the patches were removed and the remaining test item was rinsed away with sterile physiological saline solution.Twentyone hours later the skin of the animals was shorn in the region of the treatment sites.
- No. of exposures: 1
- Day(s) of challenge: 35
- Exposure period: 6 h
- Test groups: Hypoallergic patches loaded with the test item formulation were applied and fixed in place on the skin with a ORABAND self-adhesive tape.
- Control group: As a control, a patch loaded only with the vehicle was applied and fixed also to the right flank, cranial to the test item patch.
- Site: right flank (in test groups), right flank, cranial to the test item patch (control)
- Concentrations: 3 %
- Evaluation (hr after challenge): 30 and 54 h

OTHER:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated in sterile physiological saline solution to yield a solution (1% - 3%) or an emulsion (6% - 50%).
The emulsions were continuously homogenized on a magnetic stirrer during the treatments.
The stability and homogeneity of the test item formulations in the vehicle were analytically verified for up to 2 hours.

GRADING:
0 = No reaction
1 = Slight localized redness
2 = Moderate confluent redness
3 = Severe redness and swelling

EVALUATION:
The criterion for existence of sensitization was taken to be the occurrence of skin reactions in the test item group animals at a higher incidence and greater intensity than in the control animals. A test item is interpreted to be sensitizing if by comparison with the control group 15% or more of the test group animals reacted positive.

Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
alpha hexyl cinnamic aldehyde formulated in polyethylene glycol 400. Epicutaneous inductions were performed using a 40% test item formulation and challenge with 20%

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
30
Group:
test group
Dose level:
3 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 30.0. Group: test group. Dose level: 3 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
54
Group:
test group
Dose level:
3 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 54.0. Group: test group. Dose level: 3 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
30
Group:
negative control
Dose level:
3 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 30.0. Group: negative control. Dose level: 3 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
54
Group:
negative control
Dose level:
3 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 54.0. Group: negative control. Dose level: 3 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
30
Group:
positive control
Dose level:
20 %
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
40 % of the animals exhibit skin effects (grade 1).
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 30.0. Group: positive control. Dose level: 20 %. No with. + reactions: 8.0. Total no. in groups: 20.0. Clinical observations: 40 % of the animals exhibit skin effects (grade 1). .

Any other information on results incl. tables

After the first induction with 6% test item formulation 6 of 20 animals of the test item group showed severe skin effects (grey-black hardened area with red surrounding on the treatment area, size up to 1 * 0.5 cm, like corroded) and 2 of 20 animals revealed skin redness (grade 1). There were no skin effects in the animal of the control group.

After the second and third induction with 3% test item formulation no skin effects in the animals of the test item group and control group were observed.

The 1st induction was carried out on the left flank with 6% test item formulation, the 2nd and 3rd induction on the right flank with 3% test item concentration because of the strong skin effects mentioned after the 1st induction.

The skin effects mentioned were most probably due to corrosive properties of the test item.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information