Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
updated 06 July 2012
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Test material form:
Specific details on test material used for the study:
purity: 98.0 %

In vivo test system

Test animals

Details on test animals and environmental conditions:
Source: Envigo RMS B.V., Inc (Postbus 6174, 5960 AD Horst / The Netherlands)
Age (beginning of treatment): Pre-test: 11 - 12 weeks
Main study: 10 - 11 weeks
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimation: 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 the study.

Study design: in vivo (LLNA)

25, 50 and 100 % (w/v)
No. of animals per dose:
Details on study design:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25, 50, and 100% in water-free DMF. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.2 µCi of 3H-methyl thymidine (equivalent to 80.6 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm 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 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
All calculations conducted on the DPM values and the ear weights were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program a statistical analysis was conducted on the ear weights to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers.

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2016.

In vivo (LLNA)

Resultsopen allclose all
Key result
Remarks on result:
other: 25 % Dihydrolinalool: S.I. = 2.2 50 % Dihydrolinalool: S.I. = 2.9 100 % Dihydrolinalool: S.I. = 2.8 Vehicle control group (water-free DMF): S.I. = 1.0
other: Desintegrations per minute (DPM)
Remarks on result:
other: 25% Dihydrolinalool: DPM = 3403 50% Dihydrolinalool: DPM = 4499 100% Dihydrolinalool: DPM = 4221 Vehicle Control Group: DPM = 1528

Any other information on results incl. tables

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test item Dihydrolinalool was not a skin sensitiser under the test conditions of this study.
Executive summary:

In the study the test item Dihydrolinalool formulated in water-free dimethylformamide (DMF) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 25, 50, and 100% (w/v).

Clinical signs in the main experiment included squinted or closed eyes, mainly up to 1 h after application, as well as a very mild to moderate (score 1-2) erythema of the ear skin, and scaly ears in all animals. A statistically significant increase in ear weights was observed in the high dose group. This was considered not to be biologically relevant, since all S.I. were below the threshold index of 3.

In this study Stimulation Indices (S.I.) of 2.2, 2.9, and 2.8 were determined with the test item at concentrations of 25, 50, and 100% in water-free DMF, respectively.


The test item Dihydrolinalool wasnot a skin sensitiserunder the test conditions of this study.