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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23rd June 2005 - 8th November 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
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): Hexyl Salicylate
- Physical state: Colourless liquid
- Analytical purity:98.5%
- Lot/batch No.:2005146-0012
- Expiration date of the lot/batch: 26 May 2009
- Storage condition of test material: ambient temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK Limited
- Age at study initiation: 8-12 weeks
- Housing: 4 mice per cage
- Diet (e.g. ad libitum): RM1 ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%):30-70
- Air changes (per hr): minimum 15
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: From: 29 June 2005 To: 8th November 2005

Study design: in vivo (non-LLNA)

No. of animals per dose:
Not relevant

Study design: in vivo (LLNA)

Vehicle:
other: 1:3 ethanol:diethylphthalate
Concentration:
1, 2.5, 5, 10, 25 % w/v
No. of animals per dose:
4
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test substance was applied to the dorsal surface of each ear. This was repeated for three days. Three days after the third application, all the animals were injected, via the tail vein with approximately 250ul of phosphate buffered saline containing approximately 20uCi of a 2.0Ci/mmol specific gravity 3H-methyl thymedine.
Approximately 5 hours later, animals were sacrificed. The draining auricular lymph nodes were removed and placed in a container of PBS. A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 ml of PBS. Approximately 3 ml of 5% w/v trichloroacetic acid was added and after overnight precipitation at 4C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1ml of TCA.
The lymph node suspensions were transferred to scintillation vials and 100ml of scitillant optiphase was added prior to beta-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not required

Results and discussion

Positive control results:
The application of the positive control substance resulted in a greater than 3 fold increase in isotope incorporation at the 10 and 25% w/v concentrations. Therefore it was shown to be a sensitiser.

In vivo (LLNA)

Results
Key result
Parameter:
EC3
Value:
0.18
Cellular proliferation data / Observations:
The application of the test substance at concentration of 1, 2.5, 5, 10 and 25 % w/v in 1:3 Ethanol:DEP resulted in an isotope incorporation which was greater than 3 fold at all concentrations. Hence a repeat study comprising dose levels of 0.05, 0.25, 0.5, 1 and 2.5% w/v was conducted to determine the EC3 value. In the second study, the test substance resulted in an isotope incorporation which was greater than 3 fold at concentrations of 0.25 % and above.

Any other information on results incl. tables

The results are expressed as DPM per lymph node for each group:

0 (vehicle only): 4737DPM

1% w/v: 47194 DPM

2.5% w/v: 36531 DPM

5% w/v: 69591 DPM

10% w/v: 65268 DPM

25% w/v: 109201 DPM 

Repeat Test 0 (vehicle only): 5464 DPM

0.05% w/v: 10227DPM

0.25% w/v: 19466 DPM

0.5% w/v: 30613 DPM

1% w/v: 59186DPM

2.5% w/v: 59015 DPM

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Hexyl Salicylate in 1:3 Ethanol:DEP is a skin sensitiser under the conditions of the test with an EC3 value of 0.18% (45 µg/cm2).
Executive summary:

Hexyl salicylate was assessed for skin sensitisation potential using the mouse LLNA. The assay determined the level of T lymphocyte proliferation in the lymph nodes draining the site of chemical application, by measuring the amount of radiolabelled thymidine incorporated into the dividing cells. Hexyl salicylate in 1:3 Ethanol:DEP was a skin sensitiser under the conditions of the test with an EC3 value of 0.18% (45 µg/cm²).

According to Regulation (EC) No. 1272/2008, a classification of Category 1A skin sensitiser is warranted with the signal word warning and the hazard statement H317: May cause an allergic skin reaction.