Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-07-20 to 2011-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented guideline conform study including GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (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
Test material form:
other: liquid
Details on test material:
- Name of test material: 2-Methylpentanal

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.; Postbus 6174; 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 9 weeks (beginning of treatment)
- Weight at study initiation: 16.6 - 19.7 g
- Housing: single; Makrolon Type III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimatisation: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25,50 % (w/v) and 100 %
No. of animals per dose:
5 females (nulliparous and non-pregnant) per dose
Details on study design:
RANGE FINDING TESTS: a pre-test was performed in two animals
- Treatment: 25% and 100% by topical application once daily on three consecutive days.
- Irritation: Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value >3 was observed at any observation time and/or if an increase in ear thickness of > 25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation. On day 2 and 3, the animal treated with 100% showed an erythema of the ear skin (score 1). On day 3, the animal treated with 25% showed an erythema of the ear skin (score 1) as well. Other signs of irritation or signs of systemic toxicity were not observed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:

A test item was regarded as a sensitiser in the LLNA if the following criteria were fulfilled:

- First, that 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.

- 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:
Each test group of mice were treated by topical (epidermal) application (25 µL/ear/day) to the dorsal surface of each ear with test item concentrations once daily for three consecutive days. A further group of mice (control animals) were treated with an equivalent volume of the relevant vehicle alone. Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.9 µCi of 3HTdR were injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium. The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze. After washing two times with phosphate buffered saline the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4°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 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).

DETERMINATION OF LYMPH NODE WEIGHT AND CELL COUNT
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned in section 9.3.3 was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter. The values obtained were taken down manually.

DETERMINATION OF EAR WEIGHTS
After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test and the Student Newman Keuls test. Statistical significance was set at the first per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers.

Results and discussion

Positive control results:
alpha-Hexylcinnamaldehyde (25% in acetone:olive oil (4+1 v/v)):
Mean DPM per animal (5): 3720.5 +/- 1278.5
Stimulation Index (SI): 8.08

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: - 0% 2-Methylpentanal: SI: 1.00 - 25% 2-Methylpentanal: SI: 0.55 - 50% 2-Methylpentanal: SI: 1.24 - 100% 2-Methylpentanal: SI: 4.03
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
- 0% 2-Methylpentanal: Mean DPM per animal: 459.1 +/- 30.6 - 25% 2-Methylpentanal: Mean DPM per animal: 252.3 +/- 112.8 - 50% 2-Methylpentanal: Mean DPM per animal: 570.5 +/- 352.0 - 100% 2-Methylpentanal: Mean DPM per animal: 1847.9 * +/- 902.5 * Statistically significant increase vs. control group (p< 0.05)

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions of the present skin sensitisation study the test item shows skin sensitizing properties.