Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-10-07 to 2008-10-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V.
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation:
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for thestudy.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12

Study design: in vivo (LLNA)

Vehicle:
other: acetone:olive oil (4:1, v:v)
Concentration:
0, 10, 25, and 50 % (w/v) in acetone:olive oil (4+1)
No. of animals per dose:
4
Details on study design:
Administration and exposure:
For determination of the highest non-irritant and technically applicable test item concentration, a pretest was performed on two mice with concentrations of 25 and 50 % (w/v). The top dose of the test item is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. Four female mice were treated with different concentrations of the test item and vehicle alone by topical application at the dorsum of each ear lobe (Ieft and right) on three consecutive days. Five days after the first topical application, the mice were intravenously injected into a tail vein with radiolabelled thymidine (3H-methyl thymidine). Five hours after intravenous injection, the mice were sacrificed and the draining auricular Iymph nodes excised and pooled per group. Single cell suspensions of Iymph node cells were prepared from pooled Iymph nodes which were subsequently washed and incubated with trichloroacetic acid overni g.th The proliferative capacity of pooled Iymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

Data and Observations:
The proliferative response of Iymph node cells is expressed as the number of radioactive disintegrations per minute per Iymph node and as the ratio of 3HTdR incorporated into Iymph node cells of test Iymph nodes relative to that recorded for control Iymph nodes (stimulation index). In addition to the sensitising reactions the following observations and data were recorded: mortality/viability once daily, body weights prior to the first application and prior to necropsy, clinical signs (Iocal/systemic) daily from start to the terminaiion of in-life phase.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 1.91, 1.85, and 2.87 at concentrations of 10, 25, and 50 % in acetone:olive oil (4:1).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 5706, 10898, 10541, and 16338 at concentrations of 10, 25, and 50 % in acetone:olive oil (4:1).

Any other information on results incl. tables

Detailed results

     Measurment           Calculation  Result
  Test item conc.% (w/v)  Group DPM   DPM-BG  number of lymph nodes  DPM per lymph node  S.I.
 -  BG I  35  -  -  -  -
 -  BG II  17  -  -  -  -
 -  CG 1  5706  5680  8  710.0  
 10  TG 2  10898  10872  8 1359.0  1.91 
 25  TG 3  10541  10515  8  1314.4 1.85 
 50  TG 4  16312  16312  8  2039.0 2.87 

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S. 1. = Stimulation Index

The body weights of the animals,r ecorded prior to the 151 application and prior to necropsy were within the range commonly recorded for animals of this strain and age. In this study stimulation indices of 1.91, 1.85, and 2.87 were determined with the test item at concentrations of 10, 2 5, and 50% in vehicle, respectively. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in 3-foldor greater increase in incorporation of 3HTdR compared to the concurrent control, as indicated by the stimulation index. The estimated concentration of test item required to produce an S.1. of 3is referred to as the EC3 value.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was not a skin sensitiser under the described conditions.
Executive summary:

In the study the test item dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50 %.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. On the day of preparation one animal showed an abscess on the head. The relevance of this observation is questional, since the test item was applicated to the ears and the test item is regarded as non sensitizer.

In this study Stimulation Indices (S.I.) of 1.91, 1.85, and 2.87 were determined with the test item at concentrations of 10, 25, and 50 % in acetone:olive oil (4+1), respectively.

In conclusion, the test item was not a skin sensitiser in this assay.