Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study (OECD TG 429)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
according to §19 German Chemikaliengesetz
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): Licowax E FL
- Physical state: solid
- Stability under test conditions: at least one week in acetone/olive oil 4:1 (v/v)
- Storage condition of test material: room temperature
- Test item was extracted in acetone acetone/olive oil 4:1 (v/v) at a weight per volume ratio of 0.2 g/ml for 72 h at 37 °C

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, Horst, Netherlands
- Age at study initiation: 8-12 weeks
- Housing: individually in Macrolon cages
- Diet: standard diet, ad libitum
- Water: tap water, ad libitum


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

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, 100% extract of test item (extracted at a weight per volume ratio of 0.2 g/mL in acetone:olive oil (4:1) for 72 h at 37 °C)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Irritation: two mice with concentrations of 10, 25, 50 and 100% (non GLP pre-test)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one extract concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Three groups each of four female mice were treated with different concentrations of the test item extract by topical application at the dorsum of each ear lobe (left and right) an three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in body weight tables.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c where EC3 is the estimated extract concentration of the test item required to produce a 3- fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 an the local lymph node assay dose response plot.
The EC3 value of the test item could not be calculated, since all stimulation indices were below 3.

Results and discussion

Positive control results:
Stimulation Indices:
5%: 2.0
10%: 3.0
25%: 4.9
EC3: 9.9%

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 25%: 2.5 50%: 2.3 100%: 2.3 The EC3 could not be calculated
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Background: 0; 0.2 (mean: 0,.1) Control group: 2552.9; -Background: 2552.8 (8 lymph nodes pooled) 25%: 6382.3; -Background: 6382.2 (8 lymph nodes pooled) 50%: 5883.7; -Background: 5883.6 (8 lymph nodes pooled) 100%: 5852.0; -Background: 5851.9 (8 lymph nodes pooled)

Any other information on results incl. tables

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. The body weights of the animals, recorded prior to the 1st application and prior to necropsy was within the range commonly recorded for animals of this strain and age.

No systemic findings were observed during the study period. After the last treatment a slight reddening of the ears was observed in 2 animals treated with the undiluted extract, one animal treated with the 50% extract and two animals treated with the 25% extract. This observation is, however, due to its lack of severity not biologically relevant.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
An extract of Licowax E FL (25-100%) was not sensitising in a Local Lymph Node Assay in mice under the conditions of this study (extracted in acetone:olive oil (4:1) for 72 h at 37 °C).
Executive summary:

In the study the test item Licowax E FL extracted in acetone:olive oil (4:1) was assessed for its possible contact allergenic potential.

For this purpose the test item was extracted at a weight per volume ratio of 0.2 g/mL in acetone:olive oil (4:1) for 72 h at 37 °C. A local lymph node assay was performed using test item extract concentrations of 25, 50 and 100 % (undiluted extract).

The animals did not show any clinical signs during the course of the study and no cases of mortality observed. After the last application of the test item extract a slight reddening of the ears of treated animals was observed. This observation was, however, not relevant, since the reddening was not severe.

In this study Stimulation Indices (S.1.) of 2.5 and 2.3 were determined with the test item extract concentrations of 25, 50 and 100 %, respectively.

The extract of the test item Licowax E FL was not a skin sensitiser in this assay.