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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 2
Reference substance name:
Reference substance 001
Cas Number:
Specific details on test material used for the study:
Name: Alpha isomethyl ionone
Colourless liquid
Lot: 2004134-0008
CTL reference: Y13054/001
Expiry date: 12.09.2006
Purity: 97%
Storage conditions: Ambient temperature in the dark

In vivo test system

Test animals

Details on test animals and environmental conditions:
Female Mouse CBA/Ca/Ola/Hsd
Harlan Interfauna UK Limited, Blackthrone, Bicester, Oxonm UK
4 per group
Young adults from 8 to 12 weeks of age

4 mice per cage maximum
Temperature: 22 +/-3°C
Relative humidity 30-70%
Air: Minimum of 15 changes per hour
Light cycle: Artificial, giving 12 hours light, 12 hours dark
Diet: RM1 supplied by Special Diet Services Limited, Witham, Essex, UK
Water: automatic system ad libitum

Acclimatation 5 days prior start of dosing

Study design: in vivo (LLNA)

other: EtOH/DEP (1:3)
2.5%, 5%, 10%, 25% and 50% in EtOH:DEP (1:3)
No. of animals per dose:
4 female per dose
Details on study design:
volume of approximately 25 ul of 5 different concentrations of test material (%v/v) in a vehicle of 1:3 ethanol:diethyl phthalate was applied topically to the dorsum of each ear lobe (left and right) on three consecutive days. Three days after the third topical application all mice were injected intravenously into a tail vein with 250 ul of phosphate buffered saline (PBS) containing approximately 20 uCi of a 2.0Ci/mmol specific activity [3]H-methyl thymidine. Approximately 5 hours after the intravenous injection, the animals were sacrificed, the draining auricular lymph nodes excised and pooled per group (8 nodes per group). Single cell suspensions of lymph node cells were prepared by mechanical disintegration of pooled lymph nodes by passing through 200-mesh stainless steel gauze. Cell suspensions were then washed three times by centrifugation with approximately 10 ml of PBS. Approximately 3 ml of 5% w/v trichloroacetic acid (TCA) was added, precipitated overnight at 4° C, then cells were pelleted by centrifugation and resuspended in TCA. The proliferative capacity of pooled lymph node cells was determined by the incorporation of [3]H-methyl thymidine measured in a B-scintillation counter as disintegrations per minute (DPM). DPM per node for test material relative to controls was defined as the stimulation index (SI). Test material was regarded as a sensitizer in the LLNA if one or more concentrations of the test material elicited a 3-fold or greater increase in isotope incorporation relative to the vehicle control group (SI>/= 3.0).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The estimated concentration of the test material required to produce a 3-fold increase in the draining lymph node cell proliferative activity (EC3) was calculated by interpolating between two SI points using the following equation; EC3 = [(3 – d)/(b – d)] x (a – c) + c, where a = concentration with SI immediately above 3, b = SI of a, c = concentration with SI immediately below 3, and d = SI of c. EC3 values less than 0.1% were considered as the strongest sensitizers, the weaker sensitizers have a range of EC3 values of 1-10%, and non-sensitizers have EC3 values greater than 100% (Basketter, 2000; Gerberick, 2001).

Results and discussion

Positive control results:
The application of hexyl cinnamaldehyde at concentrations of 5%, 10%, and 25% w/v in acetone/olive oil (4:1) resulted in a greater than 3-fold increase inisotope incorporation at both 10% and 25% concentrations. Therefore, hexyl cinnamaldehyde was shown to ba a skin sensitiser, confirming the validity of the protocol used for the study.

In vivo (LLNA)

Key result
Cellular proliferation data / Observations:
see table below

Any other information on results incl. tables

Cell proliferation results:

Concentration of test substance (%w/v) number of lymph nodes assayed DPM DPM per lymph node SI
0 8 3254 407 N/A
2.5 8 1997 250 0.6
5 8 2012 252 0.6
10 8 4848 606 1.5
25 8 11008 1376 3.4
50 8 14974 1872 4.6

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
In conclusion, Alpha isomethyl ionone diluted in vehicle 1:3 EtOH:DEP is likely to be a skin sensitiser underthe conditions of the test. The EC3 value was calculated to be 21.8% w/v (5450 μg/cm2).
Executive summary:

The Local Lymph Node Assay was conducted in female mice. Each animal received a daily topical application of 2.5%, 5%, 10%, 25% or 50% (w/v) alpha-iso-methylionone in EtOH : DEP (3:1) on the dorsal surface of each ear for 3 consecutive days. Three days after the third topical application all mice were injected intravenously through the tail vein with 250 μl sterile saline (PBS) containing 20 μCi 3Hmethylthymidine (3H-thymidine). All mice were sacrificed five hours after the intravenous injection. Draining auricular lymph nodes were excised and were pooled for each experimental group. 3H-TdR incorporation was then measured by beta-scintillation counting and stimulation indices were determined for each experimental group. alpha-iso-Methylionone was considered to be a potential sensitizer under the conditions of the test with an EC3 value of 21.8% (5450 μg/cm2).