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Description of key information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
RCC Ltd, CH-4452 ltingen / Switzerland
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
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 (ordered)
- Housing: lndividual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 3433, batch no. 92/04 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

Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC.
Vehicle:
dimethyl sulphoxide
Concentration:
Pre-test: 0.5, 1, 5, 10% (w/v)
Main test: 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), DMSO and ethanol/water (7/3, v/v). DMSO was selected and used in the main test. In a non-GLP animal pretest in two mice, the test item was tested at four different concentrations: 0.5%, 1 %, 5%, and 10 % (w/v) on one ear each.

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 DMSO. The application volume, 25 µl, was spread over the entire dorsal surface (Ø ̴ 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 82.39 µCi/mL ³HTdR (equal to 20.6 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 β-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The β-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, that 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:
ln this study (RCC study number 858384) STIMULATION INDICES of 2.4, 3.6 and 11.2 were determined with Alpha-Hexylcinnamaldehyde at concontrations of 5 %, 10% and 25% (w/v), respectively, in acetone:olive oil, 4:1 (v/v).
The test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a skin sensitizer and an EC3 value of 7.5 % (w/v) was derived.
Parameter:
SI
Remarks on result:
other: Test substance concentration 2.5% (w/v): 1.2 Test substance concentration 5% (w/v): 1.6 Test substance concentration 10% (w/v): 2.2
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Control group: 595 DPM/node Test substance concentration 2.5% (w/v): 694 DPM/node Test substance concentration 5% (w/v): 945 DPM/node Test substance concentration 10% (w/v): 1296 DPM/node 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.

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

- Clinical signs: No toxic signs were observed in any animal of the control group. After the first topical application, the skin at dosing sites in all test animals showed black, persisting for a total of four days (2.5% w/v group) or for the remainder of the in-life phase of the study (5 and 10% w/v group).

- 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:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance was found to be a non-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. 10% was the highest technically applicable concentration in the vehicle. A control group of four mice was treated with the vehicle (DMSO) 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 1.2, 1.6 and 2.2 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 was found to be a non-sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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). 10% was the highest technically applicable concentration in the vehicle. A control group of four mice was treated with the vehicle (DMSO) 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 1.2, 1.6 and 2.2 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 was found to be a non-sensitizer.


Migrated from Short description of key information:
The test substance was found to be a non-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 does not need to be classified according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.