Registration Dossier

Administrative data

Description of key information

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:
2009-09-30 to 2009-10-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, adopted 2002-04-24
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2008-11-12
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS - CBA/CaOlaHsd
- Source: Harlan Nederland, Horst, The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 19.4 – 23.1 g
- Housing: group housing in Makrolon Type-II cages with standard softwood bedding.
- Diet (ad libitum): pelleted standard Kliba 3433 mouse maintenance diet (Provimi Kliba AG, Kaiseraugst/Switzerland)
- Water (ad libitum): community tap water from Itingen/Switzerland
- Acclimation period: 7 days prior to the start of the test

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Relative humidity: 30 - 70%
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethylformamide
Concentration:
5, 10 and 25% (w/v) concentration of test item
No. of animals per dose:
4 female mice
Details on study design:
RANGE FINDING TESTS:
A solubility experiment was performed. The highest test item concentration which can be technically used was a 25% (w/v) solution in DMF.
A pre-test was performed in two animals. Two mice were treated with concentrations of 25% and 10% (w/v) each in DMF on three consecutive days. In the pre-test clinical signs were recorded within 4 ± 2 hours and 24 ± 4 hours after each application as well as on the day corresponding to the day of preparation of the main experiment.
Results:
At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
The test item in the main study was assayed at 5%, 10% and 25% (w/v) in DMF. The top dose is the highest technically achievable concentration while avoiding systemic toxicity and excessive local irritation.

MAIN STUDY
Each test group of mice was treated by topical application to the dorsal surface of each ear lobe with the test item at concentrations of 5%, 10% or 25% (w/v) in DMF. The application volume of 25 μL was spread over the entire dorsal surface of each ear lobe (Ø about 8 mm) once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle.
Five days after the first topical application, all mice were administered with 250 μL of phosphate-buffered saline (PBS) containing 81.73 μCi/mL 3HTdR (equal to 20.43 μCi 3HTdR) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2.
The draining lymph nodes were rapidly excised and pooled in PBS for each experimental group. Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a gauze. After washing twice with approximately 10 mL phosphate buffered saline the lymph node cells were resuspended in approximately 3 mL 5% trichloroacetic acid and incubated at approximately + 5 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 1 mL 5% trichloroacetic acid and transferred to glass scintillation vials with 10 mL of ‘Irga-Safe Plus’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was measured on a β-scintillation counter. Background 3HTdR levels were also measured in two 1 mL aliquots of 5% trichloroacetic acid.

OBSERVATIONS
- Mortality / Viability: once daily from the start of acclimatisation to the termination of the in-life phase.
- Body weights: at the start of acclimatisation, on test day 2 (prior to the 2nd application) and on test day 6 (prior to 3HTdR injection).
- Clinical signs (local / systemic): on the day of animal delivery and once daily from test day 1 (after the 1st application) to the termination of in-life phase.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated.
Positive control results:
The S.I. of 1.0, 2.3 and 8.7 were determined with the test item dissolved in acetone/olive oil (4:1, v/v) at concentrations of 5, 10 and 25% (w/v), respectively.
The EC3 value calculated was 11.6%.
Based on the test results, the test item alpha-hexylcinnamaldehyde shows an allergenic potential when tested up to the concentration of 25% (w/v) in acetone/olive oil (4:1, v/v).
Parameter:
SI
Remarks on result:
other: 5% (w/v) concentration: 1.9 10% (w/v) concentration: 2.8 25% (w/v) concentration: 2.3
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control group: 1526 (dpm); 1485 (dpm-background) 5% (w/v) concentration: 2799 (dpm); 2758 (dpm-background) 10% (w/v) concentration: 4164 (dpm); 4123 (dpm-background) 25% (w/v) concentration: 3430 (dpm); 3389 (dpm-background)

A dose-response relationship was observed.

A calculation of the EC3 value was not performed because no test concentrations produced a S.I. of 3 or higher.

OBSERVATIONS:

- Mortality / Viability: no deaths occurred during the study period.

- Clinical signs: neither clinical signs on the ears of the animals nor systemic findings were observed during the study period.

- Body weights: the body weights of the animals, recorded at the start of acclimatisation, prior to the second application and prior to sacrifice, were within the range commonly recorded for animals of the strain and age.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is not a skin sensitizer.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not classified as skin sensitizer.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as skin sensitizer.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A standard LLNA study according to OECD 429 (key_LLNA_2009_Rel1) using a high purity sample of the test item (99.7%) showed no skin sensitisation potential. This assay was properly conducted using dimethyl formamide (DMF) as a vehicle. Test concentrations of 5%, 10% and 25% were examined; stimulation indices (SI) were 1.9, 2.8 and 2.3, respectively, and thus always below < 3. The positive control, hexyl cinnamic aldehyde, produced acceptable results and thus, the conclusion drawn from this study is that the test material lacks skin sensitising activity.

A second unconventional LLNA assay was performed (supporting_LLNA_2004_Rel2). This was a highly modified form of the standard OECD guideline 429 LLNA as described by Vohr and colleagues (Vohr et al, 2000). This non-standard LLNA is sometimes described as the ‘alternative LLNA’, or the ‘Integrated Model for the Chemical-Induced Differentiation of Skin Reactions (IMDS)’ and seeks to develop an index of sensitising activity relative to irritant activity on which decisions are based. It is important to note that attempts to validate this assay have failed, and the method is neither widely accepted nor widely used. Employing this assay a ‘positive’ response was recorded with 10% of the test material, but not with either 5% or 2.5%. In this assay immune activity indicative of skin sensitisation potential is measured as a function of increases in the weight of draining lymph nodes and induced changes in lymph node cellularity. Neither of these metrics (and in particular changes in node weight) is as sensitive as the incorporation by draining lymph node cells of tritiated thymidine that is used in the standard guideline LLNA. Further, ‘lymph nodes of the test groups’ animals had a higher weight compared with the negative control, although the individual data varied noticeably between groups’ and individual increases in lymph node weight did not always translate into increases in cellularity.

Furthermore, this substance lacks any significant skin irritant potential. On basis of the acute rabbit skin irritation study (OECD 404, section 7.3.1), the human volunteer patch tests for the assessment of irritant activity (section 7.10.2) and the human repeat insult patch tests (section 7.10.4), the test item is not irritating to skin and thus an interference with the detection of skin sensitisation potential can be excluded.

The only indication that the test material has some potential to cause skin sensitisation derives from a weak positive response obtained with 10% of the test material in a highly modified, and unvalidated version of the LLNA. However, this result can be discounted due to the failure of higher concentrations of the test material (25%) to elicit a positive response in a conventional, and well conducted, standard OECD guideline LLNA. Thus, the relatively small increases in the number of draining lymph node cells in an invalidated assay are not considered relevant for the chemicals safety assessment.

Based on the results of two skin sensitisation studies and the lack of skin irritating properties, Kimber and Basketter (2011) concluded that this substance does not have a potential for skin sensitisation. Accordingly, based on the weight of available evidence, it is concluded that the test item lacks significant skin sensitisation potential and should not be classified as skin sensitising.

References:

- Vohr H-W, Blumel J, Blotz A, Homey B and Ahr HJ (2000) An intra-laboratory validation of the Integrated Model for the Differentiation of Skin Reactions (IMDS): discrimination between (photo)allergic and (photo)irritant skin reactions in mice. Archives of Toxicology 73: 501-509.

- Basketter, D. & Kimber, I. (2011). Dihydroavenanthramide D: Review of skin sensitisation potential. Unpublished expert statement, 12.01.2011.


Migrated from Short description of key information:
not sensitising (OECD 429; GLP compliant)

Justification for selection of skin sensitisation endpoint:
GLP guideline study with the test item

Justification for classification or non-classification

Skin sensitisation

This substance does not have a potential for skin sensitisation andshould not be classified as a skin sensitiser according to Directive 67/548/EEC and Regulation (EC) No. 1272/2008.