Registration Dossier

Administrative data

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

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)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Gelb LD 6259 (No 08/0646-1)
- Physical state: solid/ sun yellow
- Analytical purity: Main component Gelb LD 6259: HPLC fingerprint at 230 and 400 nm: 87.6% and 89.1 area% , respectively. Free acid content: about 61.3 g/100g
- batch No.: 0817 VP 01
- Stability under test conditions: the stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Storage condition of test material: room temperature
- Other: the test substance was homogeneous by visual inspection, pH value: ca. 5 (undiluted test substance, moistened with water)

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 6 – 12 weeks
- Weight at study initiation: 18.0 g – 20.5 g
- Housing: single housed and identified by cage cards (Makrolon cage, type II)
- Diet (e.g. ad libitum): Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 7 days before the first test-substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 – 24°C
- Humidity (%): 30 – 70%
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Remarks:
Reason for the vehicle: propylene glycol was used as the vehicle because good homogeneity of the preparation was achieved; Form of application: suspension.
Concentration:
3%, 10% and 30% w/w preparations of the test substance in propylene glycol
No. of animals per dose:
5 females per group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the 30% preparation was the maximum technically applicable concentration.
- Irritation: the high concentration was selected based on the presence of ear irritation in a pretest using a 30% preparation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Test group Treatment Number of animals Animal No
Control group 1 Vehicle: propylene glycol 5 1-5
Test group 2 Test substance 3% in propylene glycol 5 1-5
Test group 3 Test substance 10% in propylene glycol 5 1-5
Test group 4 Test substance 30% in propylene glycol 5 1-5

- Name of test method: LLNA
- Criteria used to consider a positive response:
The increase SI of cell count by a factor of ≥ 1.5 and/or of 3H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.
If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.
The EC leading to the respective SI values were calculated by semi-logarithmical regression using all data points of the test groups because all concentrations induced effects above the cut-off SIs.
If a test substance does not elicit a biological relevant increase in cell count, 3H-thymidine incorporation but shows a clear concentration related increase in response, further investigation of the sensitization potential at higher concentrations should be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
- Form of application: epicutaneous application (simulating dermal contact with the compound which is possible to occur under practical use conditions).
- Application volume: 25 μL per ear; on study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μl of sterile saline into a tail vein.
- Site of application: dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site.

TERMINAL PROCEDURE:
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.
- Determination of ear weight: immediately after the death of each animal, the weight of the pooled punches was determined for each test group by punching a circular piece of tissue (diameter 0.8 cm) out of the apical part of each ear of all animals. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
- Preparation of cell suspension and determination of cell count: after weight determination, the pooled lymph nodes of each test group were stored in phosphate buffered saline in an icewater bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing all lymph nodes per test group through an iron mesh (mesh size 200 μm) into 40 mL of phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with in a ratio 1:500 and the cell count was determined.
- Measurement of 3Hthymidine incorporation of the lymph node cells: the remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.
Positive control substance(s):
other: A concurrent positive control (reliability check) with a known sensitizer was not included into this study, but studies using the positive control substance Alpha-Hexylcinnamaldehyde are performed twice a year in the laboratory.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The test substance induced a concentration dependent response in the auricular lymph node cell counts, which was biologically relevant (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) when applied as 3%, 10% and 30% preparations in propylene glycol. There was a concentration dependent increase in lymph node weights as well.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The increase of 3H-thymidine incorporation into the cells was concentration dependent and biologically relevant (increase above the cut off stimulation index of 3) at the above mentioned concentrations

Any other information on results incl. tables

- Ear weight

The 30% test-substance preparation caused some increase in ear weights (see figure below). The ears of the mice of test groups 2, 3 and 4 were minimally yellow discolored on study day 1, 2 and on the day of lymph node removal.

 - Body weights

The expected body weight gain was generally observed in the course of the study.

 - Other findings

The 30% test-substance preparation caused some increase in ear weights. The ears of the mice of test groups 2, 3 and 4 were minimally yellow discolored on study day 1, 2 and on the day of lymph node removal.

No further abnormalities or systemic toxicity were observed during general observation

 

Tables 1: Cell count; test group mean values and stimulation indice

 

 

Test group

 

Treatment

Cell counts

[Counts/Lymp node pair]

Stimulation index1

1

Vehicle propylene glycol

6765333

1.00

2*

3% propylene glycol

12236667

1.81

3

10% propylene glycol

13701333

2.03

4

30% propylene glycol

20946667

3.10

* Calculation on basis of 4 animals, as one animal died during 3H-thymidine injection;

 1test group x / test group 1 (vehicle control)

 

Table 2: 3H-thymidine incorporation; test group mean values and stimulation indice

 

 

Test group

 

Treatment

³H-thymidine incorporation

[DPM/Lymph Node Pair]

Stimulation index1

1

Vehicle propylene glycol

463.8

1.00

2*

3% propylene glycol

1727.7

3.73

3

10% propylene glycol

2586.8

5.58

4

30% propylene glycol

3256.2

7.02

* Calculation on basis of 4 animals, as one animal died during 3H-thymidine injection;

 1test group x / test group 1 (vehicle control)

 

Table 3: Lymph Node Weight; test group mean values and stimulation indice

 

 

Test group

 

Treatment

Lymph Node Weight

[mg/Lymph Node Pair]

Stimulation index1

1

Vehicle propylene glycol

4.3

1.00

2*

3% propylene glycol

5.8

1.34

3

10% propylene glycol

6.6

1.53

4

30% propylene glycol

9.9

2.29

* Calculation on basis of 4 animals, as one animal died during 3H-thymidine injection;

 1test group x / test group 1 (vehicle control)

 

Table 3: Ear Weight; test group mean values and stimulation indice

 

 

Test group

 

Treatment

Ear Weight

[mg/animal]

Stimulation index1

1

Vehicle propylene glycol

32.8

1.00

2*

3% propylene glycol

32.6

0.99

3

10% propylene glycol

33.3

1.02

4

30% propylene glycol

36.5

1.11

* Calculation on basis of 4 animals, as one animal died during 3H-thymidine injection;

 1test group x / test group 1 (vehicle control)

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information