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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted with a supporting substance, but according to the OECD guideline 429 and is fully GLP-compliant.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Principles of method if other than guideline:
The study was carried out according to
-OECD Guideline for the Testing of Chemicals No. 429, Skin Sensitisation: Local Lymph Node Assay, adopted April 24, 2002 and according to EC method B.42 (2004/73/EC);
-Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An Euro-pean inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005);
-Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An Euro-pean inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005).

The inter-laboratory validation of the method was conducted in nine laboratories in Europe. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products (Swissmedic) in Bern. Ear-draining lymph node weight and cell counts were used to assess lymph node cell proliferation instead of 3H-methyl thymidine incorporation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears to identify skin irritation properties of the test items. Statistical analysis was performed by the department of statistics (University Bern). Threshold values were calculated for the endpoints measured in this modification of the LLNA. It was concluded that all parameters measured have to be taken into consideration for the categorisation of compounds due to their sensitising potencies. Therefore, an assessment scheme has been developed which turned out to be of great importance to consistently assess sensitisation versus irritancy based on the data of the different parameters.

The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the inter-laboratory validation) were calculated by dividing the average absolute lymph node cell counts per group of the test item treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female

Study design: in vivo (LLNA)

Vehicle:
other: PEG300
Concentration:
0% (vehicle), 10%, 25%, 50%
No. of animals per dose:
6
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For lymph node weight: U-test according to MANN and WHITNEY. For possible concentration-response-relationship for the lymph node weight: linear regression analysis employing PEARSON's correlation coefficient. Outliers were determined according to the Nalimov test.
Ear weight determination: U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.

Results and discussion

Positive control results:
0% (vehicle) => 3733333 cells/ml = stimulation index of 1.0
positive control (HCA) => 6916667 cells/ml resulting in a stimulation index of 1.853

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 0% (vehicle) => 1.0 10% => 1.196 25 % => 1.339 50 % => 1.272
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Cells per ml 0% (vehicle) => 3733333 10% => 4466667 25 % => 5000000 50 % => 4750000

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance at concentrations of 10%, 25% or 50% (w/w) in PEG 300 did not reveal any sensitising properties in the local lymph node assay in mice.