Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-05-10 to 2007-09-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study performed according to OECD Guideline 429 (Skin Sensitization: Local Lymph Node Assay) with one deviation: The relative humidity in the animal room was between approximately 30 - 87% instead of 30 - 70%. This deviation to the study plan, however, did not affect the validity of the study.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 30 - 87% instead of 30 - 70%.
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate issued by Hessisches Ministerium für Umwelt, Germany
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): T000268
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: no data
- Physical state: solid
- Analytical purity: 100%
- Impurities (identity and concentrations): not applicable
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: BEA351
- Expiration date of the lot/batch: 2007-05-31
- Stability under test conditions: no data
- Storage condition of test material: at room temperature
- Other: solubility: < 0.010 g/L in water, 0.46 g/L in methanol, 68 g/L in dichloromethane, 8.5 g/L in acetone, 0.28 g/L in ethanol, 0.13 g/L in 2-propanol, and 24 g/L in N,N-dimethylformamide.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands (B.V. Postbus 6174. NL - 5960 AD Horst / The Netherlands)
- Age at study initiation: 7-8 weeks at the beginning of acclimation
- Weight at study initiation: 18.7 +/- 0.8 g
- Housing: individually in Makrolon Type I cages with wire mesh tops and granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: under test conditions after health examination, no data on acclimation period.


ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3
- Humidity (%): 30 - 87%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: no data To: no data

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
1, 10 and 25 % (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: no data
- Irritation: In a pre-test in two mice, test substance concentrations of 1, 5, 10, and 25% (w/v) were tested on one ear each; no irritation effects were observed at these concentrations after a triple application.
- Lymph node proliferation response: not measured


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Criteria used to consider a positive response: A test substance was regarded as a sensitizer in the LLNA if the following criteria were fulfilled: 1) exposure to at least one 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; and 2) 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:
- The test substance was placed into a volumetric flask on a tared balance and the vehicle (propylene glycol) was quantitatively added. The test substance concentrations were prepared individually. Homogeneity of the test substance in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion.
- Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1, 10, and 25% (w/v) in propylene glycol. The application volume, 25 uL, was spread over the entire dorsal surface (8 mm in diameter) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of propylene glycol (control animals).
- Five days after the first topical application, all mice were administered with 250 uL of 79.1 uCi/mL 3HTdR (corresponds to 19.8 uCi 3HTdR per mouse) by intravenous injection via a tail vein.
- Approximately five hours after treatment with 3HTdR, all mice were euthanized by intraperitoneal injection of Na-thiopental. The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 deg C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
- The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
other: alpha-hexylcinnamaldehyde in acetone:olive oil 4:1 (v/v)
Statistics:
The mean values and standard deviations were calculated in the body weight tables. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the below equation:

EC3 = (a-c) [(3-d)/(b-d)] + c

where EC3 was the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) were respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
The DPM measurements for the vehicle control, 5%, 10%, and 25% groups were 3115.01, 7516.37, 12579.80, and 11326.90, respectively.
The stimulation indices of the positive control groups were 2.43, 4.07 and 4.88 for the 5, 10 and 25% concentrations, respectively. The corresponding EC3 value was 6.7% (w/v).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The stimulation indices were below 3.0 for all test groups (1.18, 1.16 and 1.06 for the 1, 10 and 25% groups, respectively).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The DPM were 3275.29, 3850.56, 3809.06 and 3463.50 for the control, 1, 10 and 25% groups, respectively.

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 weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

The EC3 value could not be calculated, since none of the tested concentrations induced an SI greater than 3.

Applicant's summary and conclusion

Conclusions:
The test substance was not a skin sensitizer under the described conditions.
Executive summary:

not applicable