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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:
2003-09-24 to 2003-10-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): T002251
- Physical state: Solid
- Appearance: Brown powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: female mice (nulliparous and non-pregnant), CBA/CaOlaHsd; from Harlan Netherlands
- Age at beginning of acclimatization: 8-12 weeks
- Weight prior to the first application: 19.8 - 25.0 grams
- Housing: Standard Laboratory Conditions; individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz). In groups of four in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): ad libitum, pelleted standard Kliba 3433 mouse maintenance diet
- Water (e.g. ad libitum): ad libitum, community tap water from Itingen
- Acclimation period: at least 6 days, under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning.
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 fluorescent light/12 hour dark cycle with at least 8 hours music during the light period.

IN-LIFE DATES: From: 2003-10-01 To: 2003-10-08

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10 and 25 % (w/v)
No. of animals per dose:
4 females/group; 3 test groups and 1 control group
Details on study design:
RANGE FINDING TESTS:
- To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in two mice with concentrations of 1%, 5%, 10% and 25% (w/v) (pretest excluded from statement of compliance).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5%, 10% and 25% (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface (diam. ~8mm) of each ear lobe once daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
- Criteria used to consider a positive response: a test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled: first, that 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, that 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.

ADMINISTRATION OF ³H_METHYL THYMIDINE (³HTdR):
- 5 days after topical application, all mice were administered with 250 µl of 80.44 µCi/ml ³HTdR (equal to 20.1 µCi ³HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED ³HTdR
- Approximately 5 hours after treatment with ³HTdR all mice were euthanized by intraperitoneal injection of Vetanarcol (Veterinaria AG, Zürich).
- The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 ml) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
- The level of ³HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1 ml-aliuots of 5% trichloroacetic acid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated for body weights.

Results and discussion

Positive control results:
Stimulation indices of 1.5, 3.2 and 6.9 were determined with the positive control at concentrations of 5%, 10% and 25% (w/v) in acteone:olive oil, 4:1 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the stimulation index (S.I.).
The test item alpha-hexylcinnamaldehyde was found to be a skin sensitizer and an EC3 value of 9.4% (w/v) was derived.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.6
Test group / Remarks:
5% w/v; mean result of 4 animals
Parameter:
SI
Value:
0.8
Test group / Remarks:
10% w/v; mean result of 4 animals
Parameter:
SI
Value:
1
Test group / Remarks:
25% w/v; mean result of 4 animals
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
- background I: 0 dpm - background II: 2 dpm - control group 1: 3525 dpm; 3524 dpm-BG*; 441 dpm per lymph node (8 lymph nodes) - test group 2 (5% (w/v)): 5478 dpm; 5477 dpm-BG*; 685 dpm per lymph node (8 lymph nodes) - test group 3 (10% (w/v)): 2944 dpm; 2943 dpm-BG*; 368 dpm per lymph node (8 lymph nodes) - test group 4 (25% W/V)): 3536 dpm; 3535 dpm-BG*; 442 dpm per lymph node (8 lymph nodes) * The mean value was taken from the figures BGI and BGII ** Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

DETAILS ON STIMULATION INDEX CALCULATION
See results table: No dose-response was observed

EC3 CALCULATION
Calculation of the EC3 value was not done because no test concentrations produced a stimulation index (S.I.) of 3 or higher.

CLINICAL OBSERVATIONS:
- Vaibility/mortality: no deaths occurred during the study.
- Clinical signs: no test item-related clinical signs were observed in any animals of the control group, group 2 (5%) or group 3 (10%). Approximately 2 hours after the first topical application, a slight ear swelling was observed at both dosing sites in all mice of group 4 (25%), persisting for 4 days.

BODY WEIGHTS
The body weight of the animals, recorded prior to the 1st application and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Stimulation indices of 1.6, 0.8 and 1.0 were determined with the test item at concentrations of 5%, 10% and 25% (w/v) in acteone:olive oil, 4:1 (v/v).
A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the stimulation index (S.I.).
The test item T002251 was found to be a non-sensitizer when tested at up to the highest achievable concentration of 25% (w/v) in acteone:olive oil, 4:1 (v/v).