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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:
2012-10-11 to 2012-10-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 2010-07-22
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 2011-01-11
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS - CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Maasheseweg 87c, 5804 AB Venray, Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 19.2 – 26.6 g
- Housing: group housing in Makrolon Type-II cages with standard softwood bedding
- Diet (ad libitum): Teklab Global Rodent 2914C (Rezeptur 3255, GLP-Batch: 20/12, Provimi Kliba AG, 4303 Kaiseraugst/Switzerland)
- Water (ad libitum): community tap water from Itingen/Switzerland
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.9 – 21.2°C
- Relative humidity: 37.7 – 65.2%
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1.5, 6, 25 and 100% (w/v) concentration of test item

According to the guideline OECD 429, consecutive doses are normally selected from an appropriate concentration series such as 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc. The current study used the following doses: 100%, 25%, 6% und 1.5%.
The reason for this is as follows:
Experiences concerning odorous aldehydes have shown that these kinds of aldehydes are tested positive for sensitisation. Furthermore, past study results have shown that mostly the typical EC3 values are between 1% and 20%. For this reason, it was decided to test concentrations in this range in order to obtain experimental results in the area of the possible EC3 value.
No. of animals per dose:
4 female mice
Details on study design:
RANGE FINDING TESTS:
1) Compound solubility: the highest test item concentration which can be technically used was a 75% (w/w) solution in acetone:olive oil (4:1 v/v). The test item is a well pipettable liquid and it was tested neat (100%).
2) Irritation / Lymph node proliferation response:
- a pre-test was performed in two animals to determine the highest non-irritant test concentration.
- two mice were treated with concentrations of 50% (w/v) each in acetone:olive oil (4/1 v/v) and 100% on three consecutive days.
- clinical signs were recorded within approximately 1 hour and 24 ± 4 hours after each application as well as on the day corresponding to the day of preparation of the main experiment.
- an erythema scoring was performed (excessive local skin irritation: erythema score ≥ 3).
- ear thickness measurements were performed pre-dose, approximately 48 hours after each application as well as on the day corresponding to the day of lymph node preparation (excessive local skin irritation: increased ear thickness ≥ 25% (compared to pre-dose values)).
Results:
- animals did not show any signs of irritation or systemic toxicity.
- the test item in the main study was assayed at 1.5%, 6% and 25% (w/v) in acetone:olive oil (4/1 v/v) and 100%.
- the top dose is the highest technically achievable concentration while avoiding systemic toxicity and excessive local irritation.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
- each test group of mice was treated by topical application of 25 µL to the dorsal surface of each ear lobe with the test item at the different concentrations in the vehicle and undiluted once daily for three consecutive days. A further group of mice 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 79.53 μCi/mL 3HTdR (equal to 19.88 μCi 3HTdR) by intravenous injection via a tail vein.
- approximately five hours after treatment with 3HTdR all mice were euthanized.
- the draining lymph nodes were rapidly excised and pooled in PBS for each experimental group (8 lymph nodes per group).
- single cell suspensions of pooled lymph node cells were prepared.
- the level of 3HTdR incorporation was measured on a β-scintillation counter.

- Criteria used to consider a positive response:
A test item is regarded as a sensitizer if the following criteria are fulfilled:
- first, the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

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 1 (prior to the 1st application) and on test day 6 (prior to sacrifice).
- 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:
For the body weight mean values and standard deviations were calculated.

Results and discussion

Positive control results:
The S.I. of 1.1, 0.8 and 3.7 were determined with the positive control alpha-hexylcinnamaldehyde dissolved in acetone/olive oil (4:1, v/v) at concentrations of 5, 10 and 25% (w/v), respectively.
The EC3 value calculated was 21.5%.
A dose response relationship was observed.
Based on the test results, the positive control alpha-hexylcinnamaldehyde shows an allergenic potential when tested up to the concentration of 25% (w/v) in acetone/olive oil (4:1, v/v).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: SI-values: 1.5% (w/v) concentration: 0.7 6% (w/v) concentration: 1.7* 25% (w/v) concentration: 4.0* 100% (w/v) concentration: 11.5 EC3 = 17.0% * A calculation of the EC3 value was performed using the indicated concentrations.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Control group: 1832 (dpm); 1827 (dpm-background) 1.5% (w/v) concentration: 1327 (dpm); 1322 (dpm-background) 6% (w/v) concentration: 3079 (dpm); 3074 (dpm-background) 25% (w/v) concentration: 7243 (dpm); 7238 (dpm-background) 100% (w/v) concentration: 21051 (dpm); 21046 (dpm-background)

Any other information on results incl. tables

A dose-response relationship was observed.

OBSERVATIONS:

- Viability / Mortality: 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 first application and prior to sacrifice, were within the range commonly recorded for animals of the strain and age.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is expected to have sensitising properties and therefore, should be regarded as a dermal sensitiser.
According to Directive 67/548/EEC and its subsequent amendments, the test substance is classified as skin sensitizer (Xi; R43).
According to Regulation (EC) No 1272/2008 and subsequent regulations, the test item is classified as sensitising (Category 1B, H317).