Registration Dossier

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:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
RCC Ltd., Toxicology, CH-4452 ltingen / Switzerland
Type of study:
mouse local lymph node assay (LLNA)

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 40'812/A
- Purity: Approx. 75%
- Lot/batch No.: WP 3/03
- Expiration date: 23 April 2010
- Stability: Stable under storage conditions
- Storage conditions: At room temperature (range of 20 ± 3 °C), away from direct sunlight

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Test system: Mice, CBA/CaOlaHsd
- Source: Harlan Netherlands B.V., Postbus 6174, NL - 5960 AD Horst, The Netherlands
- Age at acclimatization: 8 - 12 weeks (beginning of acclimatization)
- Weight at acclimatization: 18.7 g-21.8 g (beginning of acclimatization)
- Housing: ln groups of four in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 3433, batch no. 84/02 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst), ad libitum.
- Water: Community tap water from ltingen, available ad libitum.
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Room temperature and humidity were monitored continuously and values outside of these ranges may have occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC.

Study design: in vivo (LLNA)

other: ethanol:water, 7:3 (v/v)
5, 10, 25% (w/v) in ethanol:water, 7:3 (v/v)
No. of animals per dose:
4 females/dose

Number of animals for the pre-test (non-GLP): 2 females
Details on study design:
To determine the highest non-iritant and technically applicable test item concentration, a non-GLP pretest was performed in two mice with concentrations of 1, 5, 10 and 25% (w/v).

The test item in the main study was assayed at three consecutive concentrations. The top dose is the highest technically applicable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (ethanol:water, 7:3 (v/v)) was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10, and 25% (w/v) in ethanol:water, 7:3 (v/v). The application volume, 25 µL, was spread over the entire dorsal surface (Ø - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear’s surface to prevent the loss of any of the test item applied.

³H-methyl thymidine (³HTdR) was purchased from Amersham International (Amersham product code no, TRA 310: specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application, all mice were administered with 250 µL of 79.5 µCi/mL ³HTdR (equal to 19.9 µCi ³HTdR) by intravenous injection via a tail vein.

Approximately five hours after treatment with ³HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of ³HTdR incorporation was then measured on a β-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of ³HTdR incorporated into lymph node cells of test group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentration) for either local toxicity or immunological suppression.
Positive control substance(s):
other: Alpha-Hexylcinnamaldehyde in ethanol:water 7:3 (v/v) using CBA/CaOlaHsd mice (RCC Study Number 849346 between 2003-05-21 and 2003-06-04
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation: EC3=(a-c)[(3-d)/(b-d)]+c where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in lymph node cell proliferative activity; (a,b) and (c,d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
ln this study (RCC study number 849346) STIMULATION INDICES was found to be a skin sensitizer and an EC3 value of 5.7 was determined with the test item at concentration of 25% (w/v) in ethanol:water, 7:3 (v/v).

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: Test substance concentration 5% (w/v): 1.9 Test substance concentration 10% (w/v): 3.1 Test substance concentration 25% (w/v): 2.1
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Control group: 381 DPM/node Test substance concentration 5% (w/v): 710 DPM/node Test substance concentration 10% (w/v): 1189 DPM/node Test substance concentration 25% (w/v): 801 DPM/node Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

Any other information on results incl. tables


No death occurred during the study period.


No test-related clinical signs were observed in any animals of the control group or group 2 (5%). On the second application day, a slight ear swelling was observed at both dosing sites in all mice of group 3 (10%) and group 4 (25%). Since second application day, all mice of group 4 (25%) excreted urine purple. These signs persisted for the remainder of the in-life phase of the study.


The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
Migrated information Criteria used for interpretation of results: EU
The test substance was found to be a skin sensitizer.
Executive summary:

In a GLP compliant Local Lymph Node Assay, tested according to OECD guideline 429, 4 female CBA mice per dose were treated daily with the test item at concentrations of 5, 10, and 25% (w/v) in ethanol:water, 7:3 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25% was the highest technically achievable concentration in the vehicle. A control group of four mice was treated with the vehicle (ethanol:water, 7:3 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio labelled thymidine (³H-methyl thymidine), five hours thereafter the mice were sacrificed and the draining auricular lymph nodes were excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine. Stimulation Indices (S.I.) of 1.9, 3.1, and 2.1 were determined with the test item at concentrations of 5, 10, and 25% (w/v), respectively, in ethanol:water, 7:3 (v/v). Based on the S.I. determined at the 5 and 10% concentration, the EC3 was determined to be 9.6% (w/v). Based on the described study and under the conditions reported, it is concluded that the test substance is a skin sensitizer.