Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13.09.05 to 16.11.05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): Crude Tall Oil
- Substance type: Complex mixture
- Physical state: Dark high viscous liquid
- Analytical purity: No data
- Composition of test material, percentage of components: Complex mixture
- Purity test date: No data
- Lot/batch No.: 2005-06-09, sample 4.
- Expiration date of the lot/batch: No data
- Stability under test conditions: No data
- Storage condition of test material: Room temperature <25oC, in the dark, may be used under light.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman GmbH
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 16.4-21.6 g
- Housing: Individually in Makrolon Type II cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (average)
- Humidity (%): 64.8 (average)
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 14.09.05 To: 19.09.05

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 and 50 % (v/v) equivalent to doses of 15, 37.5 and 75 mg, respectively.
No. of animals per dose:
Five
Details on study design:
RANGE FINDING TESTS:
- Compound concentration: 100 and 50%
- Irritation: No local skin irritation with either dose. There was a slight increase in ear thickness at the highest dose, but not at the lower dose.
- Lymph node proliferation response: Not measured.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: if the test substance induces a three-fold or greater increase in 3HTdR incorporation into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes, as indicated by the stimulation index, together with the consideration of dose response.


TREATMENT PREPARATION AND ADMINISTRATION: The test substance (25 µl/ear) was administered in three concentrations to the dorsal surfaces of the ears of the animals of the test substance groups. In a manner identical to that of animals in the treatment groups animals of one negative control group and one positive control group were treated with acetone/olive oil and hexyl cinnamic aldehyde, respectively. Each animal was treated for three consecutive days. Three days after the last administration the proliferation of the lymphocytes of the draining lymph nodes was measured by the determination of the amounts of incorporated 3HTdR (20 µCi of 3HTdR administered to mice via the tail vein).
Approximately 5 hours after 3HTdR injection all animals were sacrificed by carbon dioxide asphyxiation and the draining auricular lymph nodes were rapidly excised. The lymph nodes of each group were pooled in PBS. A single cell suspension (SCS) of lymph node cells (LNC) was prepared by gentle mechanical disaggregation of the pooled lymph nodes. The SCSs were then transferred into centrifuge tubes and LNC were pelleted by centrifugation. Afterwards supernatants were removed by aspiration. Then the LNC were resuspended and washed twice with PBS. After the final washing the supernatants were removed leaving just a small volume (<0.5 mL) and macromolecules were precipitated by incubation with 5 % trichloroacetic acid (TCA) at 4°C overnight. Each precipitate was pelleted by centrifugation and resuspended in 1 mL TCA. This suspension was transferred into scintillation vials containing 10 mL scintillation cocktail and 3HTdR incorporation was determined with a β-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data

Results and discussion

Positive control results:
Application of 25% HCA in AOO resulted in an SI of 5.3. This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SIs of the test substance groups were 0.9, 2.6 and 3.9 for the low, medium and high dose groups, respectively. Therefore there was a concentration related response. Negative control: 1. Positive control: 5.3
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Low, medium and high dose: 4338, 13098 and 19314. Negative control: 4955 Positive control: 26402

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
In a good quality Local Lymph Node Assay (reliability score 1) conducted according to OECD test guideline 429, and GLP, Crude Tall Oil was regarded as a skin sensitiser.
Executive summary:

Crude Tall Oil was diluted with acetone: olive oil (AOO), 4:1, v/v (10, 25 and 50% solutions) and was administered to three groups of five female CBA/Ca mice. Administration was performed epicutaneously to the dorsal surface of both ears, once per day on three consecutive days. The volume administered was 25 µl per ear. Positive (hexyl cinnamic aldehyde: HCA 25% in AOO) and negative (AOO) control substances were administered under identical conditions as the test substance. Five days after the first topical application, 3H-thymidine was intravenously administered to all mice via a tail vein. Approximately five hours later all animals were sacrificed, the draining auricular lymph nodes were excised, pooled for each group, and single cell suspensions were prepared. Then incorporation of 3H-methyl thymidine into the cells was determined and compared with the negative controls. The stimulation index (SI) was calculated as the ratio of the disintegrations per minute (DPM) of the dosed groups or of the positive control group to the DPM of the negative control group.

All animals survived until the end of the study, and no adverse effects were observed in any of the animals. Body mass gains were within the normal range for the strain, sex and age of the mice used. No skin irritating effects were observed in any of the groups. The SIs of the test substance were 0.9, 2.6 and 3.9 for the low, medium and high dose groups, respectively. The positive control group gave a SI of 5.3, thus demonstrating the validity of the test. According to the OECD test guideline a positive result should be regarded if the SI is equal to or greater than 3, together with consideration of the dose-response. Therefore it was concluded by the authors that Crude Tall Oil is sensitising to the skin.