[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 26 January 2005; Experiment end date - 09 February 2005; Study completion date - 08 April 2005.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

FAT40819/A TE

IUPAC Name:

FAT40819/A TE

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40819/A
- Purity: Approx. 77%
- Lot/batch No.: Red ROE 420 BOP 01/04
- Expiration date: 2 November 2009
- Stability: Stable under storage conditions
- Storage conditions: At room temperature (range of 20 ± 5 °C), away from direct sunlight. Stored in an exsiccator.

Specific details on test material used for the study:

Identity: FAT 40819/A
Description: Red brown powder
Batch number: Red ROE 420 BOP 01/04
Purity: approx. 77 %
Stability of test item: Stable under storage condition
Expiry date: 02 November 2009
Stability of test item dilution: Stable in PEG 300 for at least 7 days at room temperature
Storage conditions: At room temperature.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at acclimatization: 8 - 12 weeks
- Weight at study initiation: 16 g-24 g
- Housing: lndividually in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 3433, batch no. 69/04 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst), ad libitum.
- Water: Community tap water from ltingen, available ad libitum.
- Acclimation period: 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
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

2.5, 5, 10% (w/v)

No. of animals per dose:

- 4 females/dose
- Number of animals for the pre-test (non-GLP): 2 females

Details on study design:

PRE-TEST
In a non-GLP solubility pre-test, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v), ethanol/water (7/3, v/v) and dimethylsulfoxide (DMSO). A suitable vehicle (dimethylsulfoxide (DMSO)) was selected and used in the main test. In a non-GLP animal pre-test in two mice, the test item was tested at four different concentrations: 1 %, 2.5 %, 5 % and 10 % (w/v) in dimethylsulfoxide (DMSO), on one ear each. 24 hours after a single topical application, the pre-test results determined that 10 % (w/v) was the highest technically applicable concentration.

MAIN TEST
- TOPICAL APPLICATION
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 2.5 %, 5 % and 10 % (w/v) in dimethylsulfoxide (DMSO). The application volume, 25 µL, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied.

- ADMINISTRATION OF ³H-METHYL THYIMIDINE'
³H-methyl thymidine (³HTdR) was purchased from Amersham International (Amersham product code no, TRA 310: specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application, all mice were administered with 250 µL of 87.79 µCi/mL ³HTdR (equal to 21.9 µCi ³HTdR) by intravenous injection via a tail vein.

- DETERMINATION OF INCORPORATED ³HTDR
Approximately five hours after treatment with ³HTdR all mice were euthanized by inhalation of CO2(dry ice). 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 twice 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 'Irga-Safe Plus' scintillation liquid and thoroughly mixed. The level of ³HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

INTERPRETATION
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of ³HTdR incorporated into lymph node cells of test group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, 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 concentration) for either local toxicity or immunological suppression.

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

In this study (RCC Study number 858384) STIMULATION INDICES of 2.4, 3.6 and 11.2 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v), respectively, in acetone:olive oil, 4:1 (v/v). The positive control item ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potential, and an EC3 value of 7.5 % (w/v) was derived.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

7.3

Test group / Remarks:

2.5%

Results 2

Parameter:

SI

Value:

7.4

Test group / Remarks:

5%

Results 3

Parameter:

SI

Value:

12.2

Test group / Remarks:

10%

Results 4

Parameter:

EC3

Remarks on result:

other: An EC3 value could not be determined because this calculation requires an S.I. value of less than 3.

Any other information on results incl. tables

- Viability/mortality: No death occurred during the study period.

- Clinical signs: No clinical signs were observed in any animals of the control group. On the first application day, the skin and the surface of both ears of all mice of Group 2 (2.5 %), Group 3 (5 %) and Group 4 (10 %) were painted the red, persisting for the remainder of the in-life phase of the study.

- Body weights: The body weight of the animals, recorded prior to the first 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:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test substance was found to be a sensitizer.

Executive summary:

In a GLP-compliant Local Lymph Node Assay, tested according to OECD guideline 429, 4 female CBA mice per dose were treated daily with the test item at concentrations of 2.5, 5 and 10 % (w/v) in DMSO by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio labelled thymidine (³H-methyl thymidine). Approximately five hours thereafter the mice were sacrificed and the draining auricular lymph nodes were excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine. Stimulation Indices of 7.3, 7.4 and 12.2 were determined with the test item at concentrations of 2.5, 5 and 10 % (w/v) in DMSO, respectively. Based on the described study and under the conditions reported, it is concluded that the test substance is a sensitizer.