Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 20 November 2003 and 03 December 2003.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: performed according to OECD test guidelines and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : SB-322069
Description : white solid
Batch number : AS103703-054A1
Date received : 29 October 2003
Storage conditions : room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaBkl) strain mice were supplied by B & K Universal Ltd, Hull, 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 by indelible ink-marking on the tail and a
number written on a cage card. At the start of the study the animals were in the weight range of
15 to 23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid-floor polypropylene cages furnished
with softwood woodflakes. Free access to mains tap water and food (Certified Rat and Mouse
Diet (Code 5LF2) supplied by International Product Supplies Limited, Wellingborough,
Northants, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to
25°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
approximately fifteen changes per hour and the lighting was controlled by a time switch to give
twelve hours continuous light (06.00 to 18.00) 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:
dimethylformamide
Concentration:
0% (vehicle), 10, 25 and 50%
No. of animals per dose:
4 (pooled approach)
Details on study design:
Groups of four mice were treated with the test material at concentrations of 10,%, 25% or
50% w/w in dimethyl formamide. The preliminary screening test suggested that the test material
would not produce systemic toxicity or excessive local irritation at the highest suitable
concentration. The mice were treated by daily application of 25 μl of the appropriate
concentration of the test material to the dorsal surface of each ear for three consecutive days
(Days 1, 2, 3). The test material formulation was administered using an automatic micropipette
and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

Five days following the first topical application of the test material (Day 6) all mice were injected
via the tail vein with 250 μl of phosphate buffered saline containing 3H-methyl thymidine
(3HTdR: 80 μCi/ml, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total
of 20 μCi to each mouse.

Five hours following the administration of 3HTdR all mice were killed by carbon
dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and
pooled for each experimental group. For each group 1 ml of phosphate buffered saline (PBS) was
added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells
was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The
lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with
the project number and dose concentration. The lymph node cell suspension was transferred to a
10 ml centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all
remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node
cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was
resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the
pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After overnight incubation at 4°C, the precipitates
were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended
in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR
incorporation was measured by β-scintillation counting.

Results and discussion

Positive control results:
Test Material: α-HEXYLCINNAMALDEHYDE
SPL Project number: 039/656
Study dates: 10 October 2003 to 16 October 2003
Methods. Three groups, each of four animals, were treated with 50 μl (25 μl per ear) of
α-HEXYLCINNAMALDEHYDE as a solution in acetone/olive oil 4:1 at concentrations of 5%,
10% and 25% v/v. A further control group of four animals was treated with acetone/olive oil 4:1
alone.
Results. The Stimulation Index (SI) expressed as the mean radioactive incorporation for each
treatment group divided by the mean radioactive incorporation of the vehicle control group are as
follows: Stimulation Index (SI) 5%=1.76, 10%=2.78 and 25%=5.06.
Conclusion. α-HEXYLCINNAMALDEHYDE was considered to be a sensitiser under the
conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 10%=2.46, 25%=2.40 and 50%=1.54
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM/ear: dimethyl formamide=460.01, 10%=1130.27, 25%=1101.82 and 50%=709.98

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of
the test.