Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 15 June 2007 to 4 August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: the study was performed according to internationally recognised guidelines and GLP.

Data source

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

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 lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: 21.0 ± 1.0 g
- Housing: individually in disposable crystal polystyrene cages (22 cm x 8.50 cm x 8 cm)
- Diet: free access to SsniffR/M-H pelleted diet
- Water: free access to tap water (filtered using a 0.22 micron filter)
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 30 to 70%
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12hrs dark / 12 hrs light

IN-LIFE DATES: from 20 June 2007 to 30 July 2007

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
1st experiment: 0, 0.1, 1.0, 10% (w/v)
2nd experiment: 0, 0.256, 0.64, 1.6, 4, 10% (w/v)
No. of animals per dose:
4 females
Details on study design:
PRELIMINARY TESTS:
- Compound solubility: the test item was not soluble in the first recommended vehicle (acetone/olive oil (4/1, v/v)), in dimethylformamide and in methylethyl ketone. The test item was soluble in propyl glycol after sonication for 10 minutes and in dimethylsulfoxide. Propylene glycol was chosen as vehicle; a solution was obtained at the maximum concentration of 10%.
- Irritation: no data
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: by hand procedure (no additional information)
- Criteria used to consider a positive response: the test item was considered as a skin sensitizer when the SI for a dose group is ≥ 3 together with consideration of a dose-response relationship. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
The dosage form preparation at 10% in the main test was sonicated for 10 minutes. Before preparation, the vehicle (PG) was degassed by sonication for 15 minutes, then saturated with inert gas and kept under inert atmosphere for 15 minutes.
Fresh test item dosage forms were prepared extemporaneously under inert atmosphere on the morning of each application and were kept under inert gas. Test item dosage forms were used within 30 minutes/1 hour after preparation.

The test item was tested in two independent experiments as follow:
- three or five treated groups receiving the test item at different concentrations,
- one negative control group receiving the vehicle (Propylene Glycol),
- one positive control group
On days 1, 2 and 3 of each experiment, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI).
The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was exceeded in the positive control group (see tables).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
In the first experiment, a significant lymphoproliferation was noted at the concentration of 10%. In the absence of local irritation at the concentration of 10%, the significant lymphoproliferative response observed at this concentration was attributed to contact sensitization. The EC3 value for the test item calculated on the basis of this experiment is equal to 1.9%. However, this experiment did not allow to conclude about the potential of the test item to induce contact sensitization because any dose-effect response was observed. In the second experiment, a significant lymphoproliferation was noted at the concentration of 10%. In the absence of local irritation at the concentration of 10%, the significant lymphoproliferative response observed at this concentration was attributed to contact sensitization. A dose-effect response was observed. The EC3 value for the test item calculated on the basis of this experiment is equal to 7.7%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See tables

Any other information on results incl. tables

Clinical examination:

No mortality was noted in the first experiment. In the second experiment, one female (No. 56) of the positive control group was found dead on day 6. No clinical signs were observed prior to its death.

In both experiments, no clinical signs were observed.

The body weight change of treated animals was similar to that of control animals.

In both experiments, no cutaneous reactions and no noteworthy increase in ear thickness were observed in the animals of the treated groups.

Table 1: results of the 1st experiment

Group

Treatment concentration

Viability (%)

Cellularity index

Dpm per group

Dpm per node

Stimulation index

Increase in ear thickness (%)

Irritation level

EC3 value (%)

1

Vehicle

87.39

-

270.95

33.87

-

-0.98

-

 

2

Test item 0.1%

76.50

1.71

648.27

81.03

2.39

0.00

non irritant

1.9

3

Test item 1%

89.66

1.61

477.68

59.71

1.76

2.02

non irritant

4

Test item 10%

82.69

2.22

3836.17

479.52

14.16

6.06

non irritant

5

HCA 25%

93.02

7.42

9387.83

1173.48

34.65

-

-

 

Table 2: results of the 2nd experiment

Group

Treatment concentration

Viability (%)

Cellularity index

Dpm per group

Dpm per node

Stimulation index

Increase in ear thickness (%)

Irritation level

EC3 value (%)

1

Vehicle

88.07

-

698.82

87.35

-

-2.02

-

 

2

Test item 0.256%

89.02

0.76

350.65

43.83

0.50

-3.92

non irritant

7.7

3

Test item 0.64%

72.41

0.66

321.82

40.23

0.46

-5.77

non irritant

4

Test item 1.6%

74.56

0.89

696.70

87.09

1.00

-3.96

non irritant

5

Test item 4%

92.86

1.63

1294.85

161.86

1.85

0.00

non irritant

6

Test item 10%

91.24

1.84

2588.09

323.51

3.70

0.00

non irritant

7

HCA 25%

93.82

4.64

4120.10

686.68

7.86

-

-

 

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions of the study, the test item induced contact sensitization in the murine Local Lymph Node Assay.
A significant lymphoproliferation was noted at the concentration of 10%, in the absence of local irritation.
Executive summary:

In a dermal sensitization study using the murine Local Lymph Node Assay (LLNA) according to OECD guideline 429 and GLP, scored as validity 1 according to Klimisch criteria, the test item was tested in two independent experiments on female CBA/J mice. First experiment, on twenty females allocated to five groups; second experiment, on twenty-eight females allocated to seven groups:

- three or five treated groups of four animals receiving the test item at different concentrations,

- one negative control group of four animals receiving the vehicle (Propylene Glycol = PG),

- one positive control group of four animals receiving the positive control, α-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in Propylene Glycol.

In the first experiment, the test item was tested at the concentrations of 0.1, 1 and 10% and then in the second experiment at the concentrations of 0.256, 0.64, 1.6, 4 and 10%.

In each experiment, the test item, vehicle or positive control was applied over the ears (25 μL per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

 

First experiment:

No mortality and no clinical signs were observed during the study.

No cutaneous reactions and no noteworthy increase in ear thickness were observed in the animals of the treated groups.

A significant lymphoproliferation was noted at the concentration of 10%. In the absence of local irritation at this concentration, this effect was attributed to contact sensitization.

The EC3 value for the test item is equal to 1.9%. However, this experiment did not allow to conclude about the potential of the test item to induce contact sensitization because any dose-effect response was observed.

Second experiment:

No mortality related to treatment with the test item and no clinical signs were observed during the study.

No cutaneous reactions and no increase in ear thickness were observed in the animals of the treated groups.

A significant lymphoproliferation was noted at the concentration of 10%. In the absence of local irritation at this concentration, this effect was attributed to contact sensitization.

A dose-effect response was observed. The EC3 value for the test item is equal to 7.7%.

 

As all the acceptance criteria were met, this experiment was considered valid.

 

Under the experimental conditions of this study, the test item induced contact sensitization in the murine Local Lymph Node Assay.

According to the EC3 value obtained in the second experiment, the test item should be considered as a moderate sensitizer and classified as a skin sensitizer according to CLP criteria.