[No public or meaningful name is available]

Endpoint summary

Description of key information

The test substance was found to be a skin sensitizer in the Local Lymph Node Assay.

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:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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.

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

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.

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.

Parameter:

SI

Value:

7.3

Test group / Remarks:

2.5%

Parameter:

SI

Value:

7.4

Test group / Remarks:

5%

Parameter:

SI

Value:

12.2

Test group / Remarks:

10%

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.

- 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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

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 (RCC 2005). 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.

Short description of key information:
The test substance was found to be a skin sensitizer in the Local Lymph Node Assay.

Justification for selection of skin sensitisation endpoint:
Only study available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings in the skin sensitisation study, the substance needs to be classified with Skin sens 1, H317 according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.