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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
Study period:
From May to June 2001
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

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)
Version / remarks:
November, 2000
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:
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France.
- Females nulliparous and non-pregnant: 28 nulliparous and non-pregnant females
- Age at study initiation: on the first day of treatment, the animals were approximately 9 weeks old.
- Weight at study initiation: mean body weight ± standard deviation of 20.7 ± 1.2 g
- Housing: the animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust.Sawdust is analysed by the supplier for composition and contaminant levels. Identification was done by a number on the tail.
- Diet (e.g. ad libitum): all animals had free access to A04 C pelleted diet (UAR, Villemoisson, Epinay-sur-Orge, France).Each batch of food is analysed by the supplier for composition and contaminant levels.
- Water (e.g. ad libitum): all animals had free access to tapwater (filtered using a 0.22 micron filter) contained in bottles. Bacteriological and chemical analyses of water, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines) are performed regularly by external laboratories.
No contaminants are known to be present in the diet, drinking water or sawdust at levels which may be expected to interfere with or prejudice the outcome of the study.
- Acclimation period:at least 5 days beforethe beginning ofthe study.

- Temperature (°C): 22 ± 2 °C
- Humidity (%): 30 to 70 %
The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals.
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/ 12h

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
5 doses tested (0.05, 0.10, 0.25, 0.50 and 1 M).
No. of animals per dose:
Four animals per groups (7 groups: 5 groups treated with test item, one negative control and one positive control). Total animals: 28
Details on study design:
- Compound solubility: the test item was poorly soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v), the limit of solubility being approximately 1 M.


- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: the test item was considered as a skin sensitizer when the SI for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

The test item was prepared in the vehicle at the chosen concentrations. The concentrations were expressed in molar. All dosage form preparations were made freshly on the morning of administration and stored under nitrogen gas before use. Any unused material was discarded that same day.
The reactive used for the proliferation assay was [3H] methyl-thymidine (3h-TdR). Three days before the injections, the required quantity of 3H-TdR was diluted in 0.9% NaCl (20 µCi in 250 µL of 0.9% NaCl per animal). The obtained solution was stored at +4 °C and protected from light before use.

The concentrations of the test item were selected on the basis of the results of the solubility assay. 7 groups of 4 females each were analysed (vehicle, 0.05M, 0.1M, 0.25M, 0.5M, 1M and positive control hexylcinnamaldehyde).
On days 1, 2 and 3, a dose-volume of 25 μl ofthe control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed between each application.

- Clinical signs, morbidity and mortality
The animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality.
- Body weight
The animals were weighed individually on the first day of the study (day 1) and on the day of sacrifice (day 6).
- Ear thickness measurements and recording of local reactions
On days 1, 2 and 3 (before application) and on day 6 (after sacrifice), the thickness of the left ear of each animal of the vehicle control and treated groups was measured using a micrometer. No measurement of ear thickness was carried out for animals of the reference treated group. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (coloration, presence of residual test item,...) was noted.

- Intravenous injection of 3H-TdR and sampling of auricular lymphnodes
Lymph node cell proliferative responses were measured as described by Kimber, I. and Dearman R.J. (1991). On day 6, all animals of all groups received a single intravenous injection of 250 µl of 0.9%NaCl containing 20µCi of 3H-TdR,via the tail vein.Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe.

- Preparation of auricular lymph node cell suspensions and determination of proliferation
Cell suspensions were washed with 15 ml of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of Trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 ml of 5% (w/v) trichloroacetic acid (TCA) in purified water at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 ml of 5%TCA. Three ml of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting.
The results were expressed as disintegration‘s/mn (dpm) per group.
Stimulation Indices (SI) were calculated according to the following formula:
SI = dpm of tretaed group / dpm of control group.
The same calculation was made for the reference treated group.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Key result
ca. 0.71
Test group / Remarks:
1 M test item
Cellular proliferation data / Observations:
The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of 3H-TdR. The cell viability was higher than 80% in each group.
In the positive control group given HCA at the concentration of 25%, an increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 9.08) were noted. The study was therefore considered valid. In the treated groups, no noteworthy lymphoproliferation was noted with the test item at any tested concentrations.

Stimulation Indices (SI) were calculated accordingto the following formula:
SI = dpm of tretaed group / dpm of control group.

Calculation of the EC3 value (theoretical concentration of the test item resulting in a SI value of 3)was performed on the basis of a dose-effect response. As no evidence of a dose-relationship was noted, no extrapolated concentration EC3 was calculated.

- Systemic clinical signs and mortality
No mortality and no clinical signs were observed during the study.
- Local irritation
No cutaneous reactions and no increase in ear thickness were observed in the animals of the treated groups.
- Body weight
The body weight gain of the treated animals was similar to that of controls.
- Proliferation assay
No lymphoproliferation was noted at any tested concentration, while significant lymphoproliferation was observed with HCA used at 25%.

Any other information on results incl. tables


Test item


Sign of local


Stimulation Index



0.05 M




0.1 M




0.25 M




0.5 M











Applicant's summary and conclusion

Interpretation of results:
other: not classified as skin sensitizer according to the CLP Regulation (EC n.1272/2008)
The test item does not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The substance has been tested for skin sensatisation according to OECD TG 429 "Skin sensitization : Local LymphNodeAssay".

Twenty-eight female CBA/J mice were allocated to seven groups of four animals each: .

- five treated groups receiving the test item at the concentrations of 0.05, 0.10, 0.25, 0.50 and 1 M;

- one negative control group receiving the vehicle (mixture acetone/olive oil (4/1, v/v));

- one positive control group receiving the reference item, α-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%.

The test item, vehicle or reference item were applied over the ears (25µl per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

No mortality and no clinical signs were observed during the study. No cutaneous reactions and no increase in ear thickness were observed in the animals of the treated groups. No lymphoproliferation was noted at any tested concentration, while significant lymphoproliferation was observed with HCA used at 25%.

As indicated in the CLP Regulation (EC n.1272/2008) for Category 1, a stimulation index of three or more is considered a positive response in the local lymph node assay. For all the 5 concentrations tested the SI was lower than three. According to the CLP Regulation (EC n.1272/2008) no classification is required for skin sensitisation of the test item.