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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: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

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

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Fixapret CP konz., PBG = 10073662, after Concentration
- Analytical purity: 93.4%
- Lot/batch No.: 000335MCA0

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Research Model and Services, Sulzfeld, Germany
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 17.0 g - 20.0 g
- Housing:single housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12


Study design: in vivo (LLNA)

Vehicle:
other: 1% Pluroic L 92 Surfactant in highly de-ionized water
Concentration:
60 % w/w in 1% aqueous Pluronic
No. of animals per dose:
5
Details on study design:
Groups of 5 female CBA/J mice each were treated with a 60% w/w preparation of the test substance in 1% aqueous Pluronic® or with the vehicle alone. The 60% preparation was the maximum technically applicable concentration due to the sticky properties of the test substance.

The study was carried out as a limit test, using 1 test group and 1 control group. Each test animal was applied with 25 µL per ear of the testsubstance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.

Three days after the last application the mice were injected intravenously with 20 µL of ³H-thymidine in 250 µL of sterile saline into a tail vein. About 5 hours after the ³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and ³H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

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.

The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.

Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve for detecting a potential inflammatory ear swelling.

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.

A concurrent positive control (reliability check) with a known sensitizer was not included into this study. Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85% are performed twice a year in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 2.31
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: at 60% w/w test substance 2222.9 dpm

Any other information on results incl. tables

Test group

Treatment

Cell Count Stimulation Index

³H-thymidine incorporation Stimulatin Index

Lymph Node Weight Stimulation Index

Ear Weight Stimulation Index

 

1

Vehicle 1% aqueous Pluronic

 

1.00

 

1.00

 

1.00

 

1.00

 

2

60% in 1% aqueous Pluronic

 

1.24

 

2.31

 

1.08

 

1.21

No signs of systemic toxicity were noticed. 

 

When applied as 60% preparation in 1% aqueous Pluronic®, the test substance did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.

Applicant's summary and conclusion

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