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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-05-10 to 2011-06-01
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: In compliance with Guideline and GLP requirements, except no analysis of test item formulation. Test item was formulated within 2 hours of application, so stability was adequate for integrity of study.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
test formulations not analyzed
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): SD10
- Analytical purity: 96.9%
- Lot/batch No.: 9147-192-3a
- Expiration date of the lot/batch: 2012-11-04 date for reanalysis
- Storage condition of test material: Room temperature in dark

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Harlan Laboratories, UK Ltd, Oxon, UK
- Age at study initiation: 8 to 12 weeks of age
- Weight at study initiation: 15 to 23 grams
- Housing: individually in suspended solid-floor polypropylene cages with softwood wood flakes
- Diet (e.g. ad libitum): Teklad Global Rodent Diet
- Water (e.g. ad libitum): mains tap water ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 19 to 25 degrees C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 2011 May 10 To: 2011 June 1

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
vehicle only, or 10, 5, or 2.5% test article v/v in acetone/olive oil 4:1 in main test
undiluted, or 25% or 10% v/v in acetone/olive oil 4:1 in preliminary test
No. of animals per dose:
Preliminary test: three mice, one per concentration (25 ul undiluted per ear, or 25% or 10% v/v in acetone/olive oil 4:1) for one day, then surviving mice treated for two more days with either 25% or 10%,
Main test: 4 mice per concentration group (10, 5, or 2.5% test article v/v in acetone/olive oil 4:1) and vehicle control
Details on study design:
- Irritation: No signs of systemic toxicity, visual local skin irritation or irritation indictated by a 25% or greater increase in mean ear thickness was noted in the animal treated with test article at a concentration of 10% v/v in acetone/olive oil 4:1. The test article applied as undiluted or at a concentration of 25% in vehicle caused signs of toxicity such as hunched posture, lethargy, ataxia, decreased respiratory rate, splayed gait and ptosis. Bodyweight loss was noted in these animals.

- Name of test method: Local lymph node assay
- Animal assignment: random assignment
- Criteria used to consider a positive response: Proliferation response of lymph nodes expressed as number of radioactive disintegrations per minute per lymph node and as the ration of H^3TdR incorporation into lymph node cells of test nodes relative to control nodes (Stimulation index). Test substance was considered a sensitizer if at least one concentration of the test item resulted in a threefold or greater increase in H^3TdR incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION: In preliminary test, test article was applied undiluted or as a freshly prepared solution in acetone/olive oil 4:1. Test article was formulated within 2 hours of application. Test article (25 ul) was applied to the dorsal surface of each ear using an automatic pipette and spread over the surface of the ear with the tip of the pipette. Vehicle alone was applied in a similar manner to a group of control animals. In the main study, application of test article or vehicle were given on three consecutive days.
In the main study, 5 days after after the first topical application, all mice were injected with 250 ul of phosphate buffered saline containing H^3-methyl thymidine (H^3TdR: 80 uCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) for a total of 20 uCi per mouse.
Positive control substance(s):
other: alpha hexylcinnamaldehyde or phenyl acetaldehyde

Results and discussion

Positive control results:
Positve historical control data for the LLNA at this lab (from 2009 to March 22, 2011) showed acceptable positive results. The study most applicable in time, using acetone/olive oil 4:1 vehicle showed the following simulation indices:
15% alpha hexyl cinnamaldehyde Stimulation index 3.34
20% alpha hexyl cinnamaldehyde Stimulation index 7.33
25% alpha hexyl cinnamaldehyde Stimulation index 6.19

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: see Remark
Concentration Stimulation Index Result Vehicle not applicable not applicable 2.5% 13.82 Positive 5% 26.26 Positive 10% 38.37 Positive
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Concentration dpm dpm/node Vehicle 8544.78 1068.10 2.5% 118120.90 14765.11 5% 224393.50 28049.19 10% 327846.40 40980.80

Any other information on results incl. tables

Preliminary test: Animals treated with undiluted or 25% test article solution were euthanized on days 1 or 4 due to clinical signs of toxicity that exceeded moderate severity limit. No local skin irritation was noted in the animals. Ear thickness measurements increased over predose values 1.923% in animal S-2 (day 6) and 8.791% in animal S-3 (day 3).

Main study: There were no unscheduled deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Mild redness to head, neck and ears were noted in the animals of the 10% test article group. Body weight changes of test animals between days 1 and 6 were comparable to those observed in the corresponding control animals except for 2 test animals which showed greater than expected bodyweight loss (3 grams in an animal in low dose group, 4 grams in a high dose animal).

Applicant's summary and conclusion

Interpretation of results:
Migrated information
The test item was considered to be a sensitizer under conditions of the Local Lymph Node assay in mice.
According to CLP (Reg. 1907/2006 and amendments) the substance is classified as skin sensitizer category 1. According to
Dir. 67/548/EEC the substance is classified as sensitizing to skin (R43).
Executive summary:

A study was performed to assess the skin sensitization potential of the test article in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method complied with OECD Guideline No. 429 "Skin Sensitization: Local Lymph Node Assay", and Method B42 Skin Sensitization Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008. Following a preliminary test in which no clinical signs of toxicity were seen with a concentration of 10% test article in acetone/olive oil 4:1, this was chosen as the top dose for the main study. The other concentrations were 5% and 2.5% v/v in the same vehicle. Four groups of 4 animals each were treated with 25 ul per ear of vehicle or test solutions for three consecutive days. Animals were administered administered 20 uCi total dose of H^3TdR via tail vein on the 5th day following the first topical application. Animals were sacrificed 5 hours after the labelled methyl thymidine was given. The draining auricular lymph nodes from all mice per group were removed and pooled for preparation of a single cell suspension of pooled lymph node cells. After 18 hours of incubation, the cell precipitates were recovered by centrifugation, and resuspended for scintillation counting. The number of radioactive disintegrations per minute were then measured using a Beckman scintillation system. The Scintillation Index (mean radioactive incorporation for each treatment group divided by the mean incorporation of the vehicle control group) for the treatment groups were 2.5% (13.82), 5% (26.26), and 10% (38.37). As the scintillation index for each treatment group was greater than 3, the test item was considered to be a sensitizer under the conditions of the test.

As an EC3 was not derived in the study, the substance is classified as a skin sensitizer category 1 according to CLP (Reg. EC No. 1907/2006 and amendments) based on the study results.

According to Dir 67/548/EEC the substance is classified as sensitizing to skin (R43) based on the study results.