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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 March 2007 - 13 March 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This analogue information is used for read across to Allyl cinnamate

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca strain mice were supplied by Charles River UK Ltd., Manston Road, Margate, Kent, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study. At the start of the study the animals were in the weight range of 18.1 to 23.7 g, and were eight to twelve weeks old.

The animals were group housed in cages suitable for animals of this strain and weight range. Free access to mains water and food (RM1, Special Diet Services Limited, Witham, Essex, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 22 ± 3°C and 30 to 70 %, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was minimum 15 changes per hour and the lighting was controlled to give twelve hours continuous light and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:
other: 1:3 ethanol:diethylphthalate
Concentration:
Undiluted test item or the test item at concentrations of 0.5%, 1%, 2.5%, 5% or 10% in vehicle.
No. of animals per dose:
Groups of four mice were treated
Details on study design:
Preliminary Screening Test
Not performed. Dose levels for the main test were set by the sponsor in accordance with acute oral toxicity data provided.

Main Test
Test Item Administration
Groups of four mice were treated with the undiluted test item or the test item at concentrations of 0.5%, 1%, 2.5%, 5% or 10% v/v in 1:3 ethanol:diethylphthalate. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using a variable volume micropipette and spread over the dorsal surface of the ear. A further group of four mice received the vehicle alone in the same manner.

The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, 5%, 10% or 25% w/v preparation of Hexylcinnamaldehyde in acetone in olive oil (4:1) was applied, and a vehicle control group was similarly treated using acetone in olive oil (4:1) alone.

3H-Methyl Thymidine Administration
Three days after the third application, all animals were injected via the tail vein with approximately 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:20µCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed at least once daily for signs of toxicity. Animals were monitored for any signs of irritancy on the ears on days 1, 2, 3 and 6 of the study. Irritancy was scored at a visual level with the scoring scheme of none detectable, mild, moderate or severe.

Bodyweights: The bodyweight of each animals was recorded prior to dosing on Day 1 and prior to injection of 3H methyl thymidine on Day 6.

Terminal Procedures
Termination: Approximately five hours following the administration of 3HTdR all mice were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

Preparation of Single Cell Suspension: A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200 mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3mL of 5% w/v Trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1mL of TCA.

Determination of 3HTdR Incorporation: The lymph node suspensions were transferred to scintillation vials and 10mL of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.

Data evaluation:
The results are expressed as a disintegrations per minute (dpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle group to give a test:control ratio known as the stimulation index (SI), for each concentration.
The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a skin sensitiser.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.

Results and discussion

Positive control results:
The positive control item, Hexylcinnamaldehyde, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
3.1
Parameter:
SI
Value:
0.8
Remarks on result:
other: 0.5% test group
Parameter:
SI
Value:
1.3
Remarks on result:
other: 1.0% test group
Parameter:
SI
Value:
1.6
Remarks on result:
other: 2.5% test group
Parameter:
SI
Value:
7.5
Remarks on result:
other: 5% test group
Parameter:
SI
Value:
8.1
Remarks on result:
other: 10% test group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
DETAILS ON STIMULATION INDEX CALCULATION
The following SI values were derived at 0.5, 1.0, 2.5, 5, and 10%: 0.8, 1.3, 1.6, 7.5 and 8.1, respectively.
EC3 CALCULATION
There was an indication that the test item elicits a SI ≥ 3 when tested up to 25%, Allyl phenoxyacetate was considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) is 3.1%.
CLINICAL OBSERVATIONS/IRRITANCY SCORING:
No mortality occurred. The application of the test substance did not result in an increase in visual levels of irritancy to the skin on or around the ear area apart from on day 3, where there was a slight reddening to the ear skin at 5 and 10% dose groups. This had resolved by day 6.

Applicant's summary and conclusion

Interpretation of results:
other: Skin sensitiser.
Remarks:
According to Regulation (EC) No. 1272/2008 and its amendments.
Conclusions:
The test item was considered to be a sensitiser under the conditions of the test and resulted in an EC3 of 3.1%.
Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 0.5, 1, 2.5, 5 and 10% the substance showed SI values of 0.8, 1.3, 1.6, 7.5 and 8.1, respectively. An EC3 has been derived resulted in an EC3 of 3.1%. Based on the results the substance needs to be classified as skin sensitiser category 1B and shall be labelled with H317: May cause an allergic skin reaction according to Regulation (EC) No. 1272/2008 and its amendments.