Farnesol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Farnesol

EC Number:

225-004-1

EC Name:

Farnesol

Cas Number:

4602-84-0

Molecular formula:

C15H26O

IUPAC Name:

farnesol

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation:8 - 12 weeks (beginning of acclimatisation)
- Housing: Makrolon Type 1 with wire mesh top, single caging
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 to 70%
- Photoperiod (hrs dark / hrs light): 12 hour photoperiod with artificial light

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5%, 10% and 25%

No. of animals per dose:

4

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA assay
- Criteria used to consider a positive response: Stimulation Index >3

TREATMENT PREPARATION AND ADMINISTRATION:
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 5, 10 and 25% (w/v) in acetone:olive oil (4:1). The application volume, 25 ul, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

Five days after the first topical application, all mice were administered with 250 uL of 78.9 uCi/mL 3HTdR (corresponding to 19.73 uCi 3HTdR per mouse) by intravenous injection via a tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). The level of 3HTdR incorporation was measured on a ß-scintillation counter, expressed as the number of radioactive disintegrations per minute (DPM).

Mortality, bodyweights and clinical signs of toxicity were also recorded during the study.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

A statistical analysis was conducted for assessment of the dose-response relationship and the EC3 value was calculated. EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity.

Results and discussion

Positive control results:

The validation/positive control study was performed with alpha-Hexylcinnamaldehyde in acetone:olive oil, 4:1 (v/v) using CBA/CaOlaHsd mice in a separate study (April 2004). Stimulation indices at 5, 10 and 25 % (w/v) positive control concentrations were determined to be 1.9, 6.1 and 11.5, respectively. An EC3 pf 6.3% (w/v) was calculated.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

> 3

Results 2

Parameter:

SI

Value:

3.8

Test group / Remarks:

5% test item (w/v)

Results 3

Parameter:

SI

Value:

6.7

Test group / Remarks:

10% test item (w/v)

Results 4

Parameter:

SI

Value:

12.7

Test group / Remarks:

25% test item (w/v)

Results 5

Parameter:

other: disintegrations per minute (DPM)

Value:

771.6

Test group / Remarks:

5% test item (w/v)

Results 6

Parameter:

other: disintegrations per minute (DPM)

Value:

1 359.3

Test group / Remarks:

10% test item (w/v)

Results 7

Parameter:

other: disintegrations per minute (DPM)

Value:

2 568.9

Test group / Remarks:

25% test item (w/v)

Results 8

Parameter:

other: disintegrations per minute (DPM)

Value:

202.2

Test group / Remarks:

Control

Cellular proliferation data / Observations:

No mortality was observed during the study and the test item had no significant effect on bodyweight. The ears of all animals treated with the highest concentration (25%) were slightly red after the second treatment and all four treated animals had red inflamed ears after the third treatment. Two animals treated with 10% test item had slightly red ears after the last application. No systemic findings were, however, observed during the study period.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Stimulation indices at 5, 10 and 25 % (w/v) test item concentrations were determined to be 3.8, 6.7 and 12.7, respectively. The EC3 could not be calculated as the SI of lowest concentration tested was above 3.

Executive summary:

The skin sensitising potential of the test item was assessed by local lymph node assay (LLNA). Female mice were treated daily with the test item at concentrations of 5, 10 and 25% (w/v) in acetone:olive oil (4:1) by topical application to the dorsum of each ear lobe for 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 injected intravenously into a tail vein with radio-labelled thymidine. Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter. No mortality or adverse effects on bodyweight were observed during the study period. Redness of the ear was observed at higher concentrations and with repeat dosing. The stimulation indices at 5, 10 and 25 % (w/v) test item concentrations were determined to be 3.8, 6.7 and 12.7, respectively. The EC3 could not be calculated as the SI of lowest concentration tested was above 3. This study is considered to be reliable without restriction (Klimisch 1) as the study was GLP-compliant and was conducted according to OECD 429.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index, according to ECHA's Guidance on the Application of the CLP Criteria (2015, version 4.1). The EC3 was surpassed in the lowest concentration tested, therefore the test item is classified as a skin sensitiser.