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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 08-0034
- Test substance number: 08/0054-2

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage stability: The stability under storage conditions over the study period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH.
- Age at study initiation: 6 – 12 weeks
- Weight at study initiation: 17.5 g – 22.2 g
- Housing: single housing in Makrolon cage, type II
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water: Tap water ad libitum.
- Acclimation period: 14 days before the first test-substance application.

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

Study design: in vivo (LLNA)

Vehicle:
other: 1% Pluronic L 92 Surfactant in doubly distilled water
Concentration:
10%, 30% and 50%
No. of animals per dose:
5
Details on study design:
SELECTION OF DOSES/CONCENTRATIONS:
The selection of concentrations took into account available information on the chemical/physical properties and the composition of the test substance. In addition the results of a pretest with a 50% test-substance solution in 1% aqueous Pluronic were considered, which did not show increased ear weights and lymph node weights as indication of ear irritation. The 50% test-substance preparation was the maximum soluble concentration in 1% aqueous Pluronic. The following dose levels were selected: 10%, 30% and 50% in 1% aqueous Pluronic.

MAIN STUDY:
- Criteria used to consider a positive response: In order to reveal a possible induction of sensitisation, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the ear backs is determined. The parameters used to characterize the response are lymph node cell count, ³H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. Because not only sensitization induction but also irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test-substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance.
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.
In addition the evaluation uses the following considerations:
If biologically relevant increases in ear weights are running in parallel to the increase in cell count, ³H-thymidine incorporation and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitisation. Depending on the magnitude of lymph node response the evaluation of the sensitizing potential may be modified or additional studies might be necessary by expert judgement.
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:
The test-substance preparation was produced on a weight per weight basis shortly before the application. After stirring with a magnetic stirrer the test substance is soluble in the vehicle.
- Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 μL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control 1% aqueous Pluronic
Key result
Parameter:
SI
Value:
0.88
Test group / Remarks:
10% preparation of test item in 1% aqueou Pluronic
Key result
Parameter:
SI
Value:
0.97
Test group / Remarks:
30% preparation of test item in 1% aqueou Pluronic
Key result
Parameter:
SI
Value:
0.99
Test group / Remarks:
50% preparation of test item in 1% aqueou Pluronic

Any other information on results incl. tables

The stimulation indices (fold of change as compared to the vehicle control) for cell count, ³H-thymidine incorporation, lymph node weight and ear weight are summarized for each test group in the following table:  

Test group

Treatment

Cell Count Stimulation Index

³H-thymidine Incorporation Stimulation Index

Lymph Node Weight Stimulation Index

Ear Weight Stimulation Index

1

vehicle 1% aqueous Pluronic

1.00

1.00

1.00

1.00

2

10% in 1% aqueous Pluronic

0.88

1.87

1.05

0.99

3

30% in 1% aqueous Pluronic

0.97

1.70

1.19

0.99

4

50% in 1% aqueous Pluronic

0.99

1.93

1.22

1.02

 

No signs of systemic toxicity were noticed. There was no relevant increase in lymph node weights. The test-substance preparations did not cause an increase in ear weights.

ANALYSES:

- Stability of the test substance preparation: The stability of the test substance in the vehicle for the maximum application period was confirmed indirectly by analysis of the correctness of the concentration.

- Homogeneity of the test substance preparation: The test-substance preparation was visually homogeneous (solution).

- Concentration control analysis of the test substance preparation: The correctness of the concentration of the test-substance preparations (10%, 30% and 50%) for the first application was confirmed by analysis.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The substance does not show skin sensitisation in the LLNA.