Registration Dossier

Administrative data

Description of key information

A local lymph node assay with hexyl salicylate is available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
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)
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
No. of animals per dose:
Not relevant
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
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.
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.

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

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Betts (2006) reports a positive result for hexyl salicylate in a LLNA. Hexyl salicylate dissolved 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). In a published review, Belsito (2007) reports a positive result in a guinea pig (modified Draize) study with 5% hexyl salicylate.

The results of animal tests with hexyl salicylate are in contrast with the human skin sensitisation data. Two human repeat insult patch tests showed no indications of hexyl salicylate inducing a sensitisation response. In one test 104 subjects were exposed to 30% hexyl salicylate without any positive response observed. In the second study, 5/22 initial results were considered to be positive for sensitisation, however, following biopsy and histopathological examination these reactions were confirmed to indicate irritation rather than hypersensitivity reactions and it was concluded that none of the responders had shown dermal contact sensitisation. Furthermore, the occupational exposure records indicate no cases of identified human sensitisation resulting from occupational exposure to hexyl salicylate (although it is recognised that this may reflect the industry standard use of protective equipment such as gloves during manufacture or handling).

Hexyl salicylate is considered to be a skin sensitiser as a precautionary approach, based on the animal data. The results of the LLNA indicate that classification in Category 1A is appropriate. Further analysis by Roberts et al. (2015), however, contrasts the high potency of hexyl salicylate in the LLNA with the lack of evidence of skin sensitisation from human exposure or from structrural considerations. The LLNA result for hexyl salicylate is therefore considered to be anomalous. It is doubtful, therefore, whether hexyl salicylate should be classified as a skin sensitiser however classification in Category 1B is considered to be adequately conservative, taking into account the absence of reports of sensitisation in exposed humans.

David W. Roberts, Anne Marie Api, Robert J. Safford & Jon F. Lalko (2015). Principles for identification of High Potency Category Chemicals for which the Dermal Sensitisation Threshold (DST) approach should not be applied. Regulatory Toxicology & Pharmacology 72(3): 683 -693.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No evidence of local effects was seen in a 28 -day inhalation toxicity study with hexyl salicylate and there are no human data indicating skin sensitisation. Furthermore, there are no concerns relating to respiratory sensitisation based on the molecular structure of hexyl salicylate.

Justification for classification or non-classification

Based on the results of the key study (LLNA) conducted by Betts (2006), hexyl salicylate should be classified as a Category 1A skin sensitiser according Regulation (EC) No 1272/2008. However further analysis of the results of this study in conjunction with human data indicate that the result of the LLNA is spurious. Based on the weight of evidence, classification in Category 1B is considered to be more appropriate. No evidence of local effects was seen in a 28 -day inhalation toxicity study with hexyl salicylate and there are no human data indicating skin sensitisation. Furthermore, there are no concerns relating to respiratory sensitisation based on the molecular structure of hexyl salicylate. Classification for respiratory sensitisation is therefore not proposed.