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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-05-19 to 2004-05-24 (positive control); 2004-07-21 to 2004-07-27 (main experiment)
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
no certificate
Type of study:
mouse local lymph node assay (LLNA)

Test material

Details on test material:
- Name of test material (as cited in study report): Benzyl salicylate
- Substance type: Colourless liquid
- Physical state: Liquid
- Analytical purity: 99.8 %
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: 2004-11-09
- Lot No: 2004134-0010
- Expiration date of the lot: 2006-05-13
- Stability under test conditions: No data
- Storage condition of test material: Ambient temperature in the dark

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Harlan Interfauna Ltd, Blackthorne, Bicester, Oxon, United Kingdom
- Age at study initiation: Young adults (8 - 12 wks)
- Weight at study initiation: 16.8 g to 21.1 g
- Housing: ≤ 4 mice per cage, in suitable cages for this strain and weight range.
- Diet: Ad libitum RM1, Special Diet Services, Witham Essex, United Kingdom.
- Water: ad libitum mains water.
- Acclimation period: ≥ 5 d

- Temperature: 22 °C ± 3 °C
- Humidity: 30 % to 70 %
- Air changes (per hr): ≥ 15
- Photoperiod: 12 hrs dark / 12 hrs light

Study design: in vivo (LLNA)

other: Ethanol:diethylphthalate (1:3)
2.5 % w/v, 5 % w/v, 10 % w/v, 25 % w/v and 50% w/v (No correction was made for the purity of the active ingredient of the test substance).
No. of animals per dose:
4 animals per dose
Details on study design:
Approximately 25 µL of preparation of the relevant concentration of test substance/vehicle was using a variable volume micropipette to the dorsal surface of each ear. A vehicle control group was similarly treated using vehicle alone. This procedure was repeated daily for 3 consecutive days.

3 d after the 3rd application, all the animals were injected, via the tail vein, with 250 µL of phosphate buffered saline (PBS) containing 25 µCi of a 2.0 Ci/mmol specific activity ³H-methyl thymidine. Approximately 5 hrs later, the animals were humanely killed by inhalation of halothene vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

A single cell suspension was prepared by mechanical disintegration of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were washed 3 times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5 % w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4 °C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA.

The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Octiphase) was added prior to β-scintillation using a Packard Tri-Car Liquid scintillation counter.

Animals were checked at least daily for signs of systemic toxicity.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The EC₃ value was derived by interpolating between two points on the stimulation index axis, using values immediately above and below the value of 3. The equation is as follows:

EC₃ = (3 - d) × (a - c) ÷ (b - d) + c

a = The concentration giving the SI immediately above 3
b = The SI value of a
c = The concentration giving the SI immediately below 3
d = The SI value of c

From this value a value of quantity applied per square centimetre was derived, assuming that the area of the mouse ear is 1 cm² and that 1 µL is equivalent to 1 mg.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: 2.5 % - 2.6; 5 % - 5.5; 10 % - 6.0; 25 % - 18.9; 50 % - 26.2 For full results table please refer to the attached supporting information.
other: disintegrations per minute (DPM)
Remarks on result:
other: 0 % (vehicle control) - 2248 DPM; 2.5 % - 5728 DPM; 5 % - 12419 DPM; 10 % - 13518 DPM; 25 % - 42548 DPM; 50 % - 58830 DPM. For full results table please refer to the attached supporting information.

Any other information on results incl. tables

Evaluation of the test substance

The EC₃ value was derived as described above and determined to be 2.9 %

From this, the quantity applied per square centimetre was determined to be 725 µg/cm.

Body weights

Information on body weights is given in the attached supporting information.

Positive control

The application of hexylcinnamaldehyde at concentrations of 5 %, 10 % and 25 % w/v in acetone:olive oil (4:1) resulted in a greater than threefold increase in isotope incorporation at both 10 % and 25 %. Thus, hexylcinnamaldehyde was shown to be a potential skin sensitiser under the conditions of the test.

Applicant's summary and conclusion

Interpretation of results:
Migrated information Criteria used for interpretation of results: EU
Benzyl salicylate was shown to be a skin sensitiser in a local lymph node assay according to OECD 429.