Registration Dossier

Administrative data

Description of key information

A modern, guideline-compliant mouse LLNA is avai;abe for amyl cinnamic aldehyde

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:
1 December 2005 to 7 April 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline compliant and GLP study with no deficiencies
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca/Ola/Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK Ltd
- Age at study initiation: 8-12 weeks
- Weight at study initiation:
- Housing: groups of four per cage
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-3°C
- Humidity (%):30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 December 2005 To: 13 December 2005
Vehicle:
other: 1:3 ethanol:diethylphthalate and 4:1 acetone:olive oil for the positive control
Concentration:
1, 2.5, 5, 10 or 25% w/v
No. of animals per dose:
Four females
Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: One or more concentration should elicit a 3-fold or greater increase in isotope incorporation.

TREATMENT PREPARATION AND ADMINISTRATION: The mice were acclimatised for a period of 5 days prior to dosing. Groups of four females were allocated to each group. Alpha amyl-cinnamaldehyde was formulated in 1:3 EtOH:DEP and administered by painting the dorsal surface of each ear with25 µl of 1, 2.5, 5, 10 or 25 % w/v on three consecutive days. Three days after the last application all animals were injected via the tail vein with 250 µl of phosphate buffered saline containing 20µCi of a 2.0 Ci/mmol specific activity tritiated methylthymidine. After five hours the mice were terminated and draining auricular lymph nodes were removed from each mouse. The nodes were pooled per group. A single cell suspension was prepared from the disaggregated lymph nodes. After preparation the lymph node suspensions were transferred to scintillation vials and Optiphase scintillant was added prior to counting with a liquid scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value was calculated from the standard formula EC3 = [(3-d)/(b-d)] x (a-c) +c by interpolating between two points on the Stimulation Index axis.
The quantity applied per square centimetre was derived from the EC3 value assuming the area of the mouse ear equates to 1 cm2 and that 1µl is equivalent to 1 mg.
Positive control results:
A positive response was shown to HCA at concentrations of 10% (SI = 4.2) and 25% w/v (SI = 15.1), confirming the sensitivity of the assay.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
A greater than three-fold increase in Stimulation Index was evident at the 10 and 25% concentrations. The concentration causing a 3-fold increase in lymphocyte proliferation (EC3) was calculated to be 7.6% w/v or 1900 µg/cm2. The positive control , hexylcinnamaldehyde, administered at three concentrations, 5, 10 and 25% w/v in acetone/olive oil resulted in a 3-fold increase in lymphocyte proliferation.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 3029, 5191, 4932, 5338, 12431 and 30873 dpm for the vehicle control and groups dosed at 1, 2.5, 5, 10 and 25 % w/v respectively.

SI Values

Substance

Concentration (w/v)

SI

Amyl cinnamic aldehyde

0 (vehicle)

-

1%

1.7

2.5%

1.6

5%

1.8

10%

4.1

25%

10.2

Hexyl cinnamic aldehyde

0

-

5%

2.3

10%

4.2

25%

15.1

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Alpha-amyl cinnamaldehyde was a skin sensitiser under the conditions of the LLNA, with an EC3 value of 7.6% w/v or 1900 µg/cm2.
Executive summary:

Alpha-amyl cinnamaldehyde was assessed for sensitising potential in the murine LLNA . Dose levels of 1, 2.5, 5, 10 and 25% w/v were applied in 1:3 ethanol:diethylphthalate. The two higher doses, 10 and 25% w/v caused an increase in lymphocyte proliferation that was greater than 3 -fold compared with the vehicle control. The EC3 value was 7.6% w/v or 1900 µg/cm2.

Alpha-amyl cinnamaldehyde was found to be a potential sensitiser under the conditions of this study and would require classification

in sub-Category 1B according to the 2nd ATP of the CLP Regulation

1272/2008

. .

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A modern, guideline-compliant mouse LLNA reports a positive response to amyl cinnamic aldehyde at concentrations of 10 and 25% w/v; an EC3 value of 7.6% is calculated.

The skin sensitisation potential identified in mice is not substantiated in human volunteer RIPT investigations where only slight transient and non-specific responses were observed and the conclusion for each of 7 assays in humans was that no evidence for skin sensitisation was found in humans. However the similarity of ACA to HCA which is used as a positive control for dermal sensitisation assays, indicates the LLNA result should be considered valid, rather than a false positive, and predictive of sensitising potential for cinnamic aldehydes, even if this is only expressed in sensitive human individuals.

Migrated from Short description of key information:

Determination of sensitising potential as indicated by lymphocyte proliferation in a murine local lymph node assay.

Additional supporting information is presented in reports of human exposure.

Justification for selection of skin sensitisation endpoint:

Positive result reported

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The results of the mouse LLNA trigger classification of amyl cinnamic aldehyde as a skin sensitiser: the EC3 value of 7.6% from this study indicates classification in sub-Category 1B according to the 2nd ATP of the CLP Regulation.