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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 December 2013 - 05 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

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 lymph node assay (LLNA)

Test material

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

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approximately 8 weeks old on the day of treatment
- Mean body weight at study initiation: the animals of the preliminary test had a mean body weight of 18.4 g (range: 18.2 g to 18.6 g) and the animals of the main test had a mean body weight of 20.4 g (range: 18.8 g to 22.5 g).
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 6 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 22 January 2014 to 03 February 2014

Study design: in vivo (LLNA)

Vehicle:
other: dimethylformamide (DMF)
Concentration:
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
- the vehicle was selected on the basis of producing a homogeneous preparation suitable for application of the test item,
- the concentrations were selected from the concentration series 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
- the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an = 25% increase of the ear thickness.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
- the vehicle was selected on the basis of producing a homogeneous preparation suitable for application of the test item,
- the concentrations were selected from the concentration series 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
- the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an = 25% increase of the ear thickness.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness and radioactivity levels.

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in the vehicle.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
On days 1, 2 and 3, at approximately the same time each day, a dose-volume of 25 µL of the control or dose formulation preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
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.
Positive control substance(s):
other: a-hexyl cinnamaldehyde (HCA)

Results and discussion

Positive control results:
SI = 7.42

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
50 % EKKE
Value:
1.64
Key result
Parameter:
SI
Remarks:
25% EEKE
Value:
1.15
Test group / Remarks:
EKKE
Key result
Parameter:
SI
Remarks:
10% EKKE
Value:
2.05
Key result
Parameter:
SI
Remarks:
5% EKKE
Value:
1.5
Key result
Parameter:
SI
Remarks:
2.5 % EKKE
Value:
1.33
Key result
Parameter:
SI
Value:
7.42
Test group / Remarks:
Positive control

Any other information on results incl. tables

The results are presented in the following table:

 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

2.5

I

1.33

Test item

5

I

1.50

Test item

10

I

2.05

Test item

25

I

1.15

Test item

50

I

1.64

HCA

25

-

7.42

-: not recorded.

I: non-irritant (increase in ear thickness < 10%).

HCA: a-hexyl cinnamaldehyde.

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
EKKE, tested as a homogeneous suspension at concentrations of 2.5, 5, 10, 25 and 50% in dimethylformamide, gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.
Therefore, the test item should not be classified as a skin sensitizer according to the criteria of CLP Regulation.
Executive summary:

The potential of EKKE to induce contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). This study was conducted in compliance with OECD Guideline No. 429 and the principles of Good Laboratory Practices. To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 5, 10, 25 or 50% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. On completion of the observation period, the animals were sacrificed then discarded without macroscopicpost-mortemexamination. In the main test, five groups of four female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 2.5, 5, 10, 25 or 50% under a dose-volume of 25 µL. One negative control group of four females received the vehicle dimethylformamide (DMF) under the same experimental conditions. Additionally, one positive control group of four females received the positive control,a-hexylcinnamaldehyde (HCA), at 25%in a mixture Acetone/Olive Oil (4/1; v/v)under the same experimental conditions.From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of3H-TdR. The results are expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

No unscheduled deaths and no clinical signs were observed during the observation period. Body weight of animals was unaffected by the test item treatment. No local reactions were observed in any animals. In all test item-treated groups, the increase in ear thickness was below 10%. The threshold positive value of 3 for the SI was reached in the positive control group (SI = 7.42). The experiment was therefore considered valid. No significant lymphoproliferation was noted with the test item at any tested concentrations as all SI values were below the cut-off value of 3.

 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

EKKE

2.5

I

1.33

EKKE

5

I

1.50

EKKE

10

I

2.05

EKKE

25

I

1.15

EKKE

50

I

1.64

HCA

25

-

7.42

-: not recorded.

I: non-irritant (increase in ear thickness < 10%).

 

EKKE gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties. Therefore, EKKE not be classified as a skin sensitizer according to the CLP and GHS criteria.