Registration Dossier

Administrative data

Description of key information

The test substance does not show a skin sensitising potential in the Local Lymph Node Assay (OECD 429, GLP).

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:
01 Jun 2004 to 15 Jul 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
April 24, 2002
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March, 2003
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Specific details on test material used for the study:
TEST MATERIAL
- Name of the test substance: DKDS - Reinkristallisat
- Batch No.: CP 203-362-04-02
- Appearance: Solid, powder / white
- Purity: 96.8 w/w%
- Homogeneity: The test substance was homogeneous by visual inspection.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. The stability under storage conditions was confirmed by reanalysis.
Species:
mouse
Strain:
CBA
Remarks:
CaOIaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, FRG
- Age at study initiation: About 6 - 8 weeks
- Weight at study initiation: 16.8 - 20.5 g
- Housing: One animal per cage (type: Makrolon 1)
- Bedding: Granulat Typ ¾ (staubfrei); SSNIFF
- Diet: Kliba-Labordiät (Maus / Ratte Haltung "GLP"), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 15 days before the first test substance application.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Photoperiod: 12/12 h
Vehicle:
other: Acetone
Concentration:
- Control group: vehicle alone
- Test group 2: 25 µL of 3% in acetone
-Test group 3: 25 µL of 10% in acetone
- Test group 4: 25 µL of 30% in acetone

The selection of concentrations took into account available information on the chemical/physical properties and the composition of the test substance, the acute toxicity and on primary irritation/corrosion potential.
No. of animals per dose:
6
Details on study design:
TEST SUBSTANCE PREPARATION
The test substance preparations were produced on a weight by weight (w/w) basis shortly before the application by stirring with a magnetic stirrer. The test substance was applied as a solution for all applications. the highest test substance concentration (30%) was found to be the maximum soluble concentration. The 30% preparation was soluble after stirring tor a prolonged period, only. Suspensions would have posed difficulties concerning homogeneity and application.

GENERAL
The study comprised three treatment groups and a vehicle control group. Each group consisted of 6 mice. A check for dead or moribund animals was made twice each workday and once on Saturdays, Sundays and on public holidays. No detailed clinical examination of the individual animais was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data.

BODY WEIGHT
Body weights of the individual animals were determined on study day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.

TEST SUBSTANCE APPLICATION
Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 µL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 - day 2) to the same application site
The animals of groups 1-4 were treated with vehicle or test substance preparation.

TERMINAL PROCEDURES
The animals were sacrificed on study day 5 by cervical dislocation

EAR WEIGHT
Immediately after the death of each animal a circular piece of tissue (diameter of 0.8 cm) was punched out of the apical part of each ear. The weight of the pooled punches was determined for each animal.

LYMPH NODE WEIGHT
Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled Iymph nodes from both sides was determined for each animal.

PREPARATION OF CELL SUSPENSIO AND DETERMINATION OF CELL COUNT
After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline in an ice-water bath until further preparation. A single ceIl suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into 6 mL of phosphate buffered saline. For determination of cell count the standard suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter. Each suspension was measured in triplicate (3 dilutions of the standard suspension). The mean of the triplicate measurements was used for further evaluation.
Positive control substance(s):
other: Alpha-Hexylcinnamaldehyde, techn. 85%
Statistics:
The statistical evaluations were performed using the WILCOXON test.
Positive control results:
A concurrent positive control (reliability check) with a known sensitizer was not included into this study.
No signs of systemic toxicity were noticed. The positive control substance induced a statistically significant and biologically relevant response in the auricular lymph nodes, when applied as 3% or 10% preparations in acetone. The Iimited but statistically significant increases in ear weights indicate some irritation of the ear skin at all concentrations.
The increase in cell count index induced by the 3% test substance preparation was at the border of biological significance. The marked increase at 10% cannot be explained by skin irritation alone.
Based on the results discussed above it is concluded that Alpha-Hexylcinnamaldehyde, techn. 85% has a sensitizing effeot in the Murine Local Lymph Node Assay under the test conditions chosen.
The threshold of skinsensitization induction of the compound is >1% <3% under the test conditions used.

Parameter:
other: Lymph node weight index
Value:
0.93
Test group / Remarks:
3% in acetone
Parameter:
other: Lymph node weight index
Value:
1.06
Test group / Remarks:
10% in acetone
Parameter:
other: Lymph node weight index
Value:
1.15
Test group / Remarks:
30% in acetone
Parameter:
other: Cell Count Index
Value:
0.99
Test group / Remarks:
3% in acetone
Parameter:
other: Cell Count Index
Value:
1.13
Test group / Remarks:
10% in acetone
Parameter:
other: Cell Count Index
Value:
1.24
Test group / Remarks:
30% in acetone
Parameter:
other: Ear Weight Index
Value:
1
Test group / Remarks:
3% in acetone
Parameter:
other: Ear Weight Index
Value:
1.02
Test group / Remarks:
10% in acetone
Parameter:
other: Ear Weight Index
Value:
0.99
Test group / Remarks:
30% in acetone
Cellular proliferation data / Observations:
LYMPH NODE WEIGHTS AND CELL COUNTS
Treatment of the mice with a 30% test substance preparation induced a slight statistically significant increase in Iymph node ceII counts but not in Iymph node weights as compared to the vehicle control group. Neither the 3% nor the 10% test substance solution caused any statistically significant Iymph node response.

EAR WEIGHTS
Treatment of the mice with 3% - 30% test substance preparations induced no statistically significant increase in ear weights as compared to the vehicle control group.

BODY WEIGHTS
The expected body weight gain was generally observed in the course of the study

OTHER FINDINGS
Rests of test substance were noted in the animals treated with the 30% test substance concentration on the day of the 2nd and 3rd application.
Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

LLNA

The skin sensitizing potential of the test item was assessed in vivo using the non-radioactive variant of the Murine Local Lymph Node Assay, an LLNA test. The study was performed according to OECD 429, adopted in 2002, under GLP conditions (BASF, 2004). Groups of 6 female CBA/Ca mice each were treated with 3%, 10% and 30% w/w preparations of the test substance in acetone or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was applied with 25 µL per ear of the respective test substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone. Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content (indicator of cell proliferation) and weight of each animal's pooled lymph nodes. Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed. At 30%, the test substance induced a slight statistically significant increase of the cell counts, but not in the lymph node weights, this effect was therefore considered not biologically relevant. No other significant effects were noted. The ear weight did not indicate an irritant property of the test substance at all concentrations. Based on these results, it was concluded that the test substance does not have a skin sensitizing effect under the test conditions chosen.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The stimulation indices in the LLNA (OECD 429) did not show a dose dependent increase. No EC3 could be established. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.