Registration Dossier

Diss Factsheets

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-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)
Version / remarks:
(adopted 24 April 2002)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-diamino-9,9',10,10'-tetrahydro-9,9',10,10'-tetraoxo[1,1'-bianthracene]-3,3'-disulphonate
EC Number:
EC Name:
Disodium 4,4'-diamino-9,9',10,10'-tetrahydro-9,9',10,10'-tetraoxo[1,1'-bianthracene]-3,3'-disulphonate
Cas Number:
Molecular formula:
disodium 4,4'-diamino-9,9',10,10'-tetraoxo-9,9',10,10'-tetrahydro-1,1'-bianthracene-3,3'-disulfonate
Details on test material:
- Physical state: Red solid
- Lot/batch No.: 00584CL6
- Expiration date of the lot/batch: 01-APR-2011
- Stability: Stable under storage conditions
- Storage condition of test material: At room temperature (range of 20 ± 5 °C), light protected.

In vivo test system

Test animals

other: CBA/CaHsdRcc(SPF)
Details on test animals and environmental conditions:
- Source: RCC Ltd, Laboratory Animal Service, Fuellinsdorf / Switzerland
- Age at study initiation: 11-12 weeks (beginning of acclimatization)
- Weight at study initiation: 18 g - 20.5 g (at delivery)
- Housing: individually
- Diet: Pelleted standard Kliba 3433, batch no. 001/06 mouse maintenance diet (Provimi Kliba AG, Kaiseraugst) available ad libitum.
- Water: Community tap water from Itingen, available ad libitum.
- Acclimation period: 7 days prior to the first topical application under test conditions after health examination. (21.06.2006 - 27.06.2006)

- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12 (at least 8 hours music during the light period)

Study design: in vivo (LLNA)

1, 2.5, 5%
No. of animals per dose:
4 female animals (3 females for the non-GLP pre-test)
Details on study design:
In a non-GLP solubility pre-test, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v), ethanol/water (7/3, v/v) and DMF. DMF was found to be a suitable vehicle and was selected and used in the main test. 5% (w/v) was the highest technically achievable concentration in the chosen vehicle. A non-GLP local toxicity pre-test was performed for determination of concentrations for the main test. Three single animals were each treated with one of three different concentrations: 1%, 2.5% and 5% (w/v), on both ears on three consecutive days. One day after each topical application, the red staining produced by the test item was found at all the dosing sites. One day after the third topical application, a slight ear swelling was observed at the dosing of 2.5% and 5%. 5% (w/v) was the highest dosing concentration in the main test.


Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with the test item at concentrations of 1%, 2.5% or 5% (w/v) in DMF. The application volume, 25 µl, was spread over the entire dorsal surface (diameter ~ 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).

Five days after the first topical application, all mice were administered with 250 µl of phosphate-buffered saline (PBS) containing 81.94 µCi/ml 3HTdR (equal to 20.4 µCi 3HTdR) by intravenous injection via a tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2 (dry ice). The draining lymph nodes were rapidly excised and pooled in PBS for each experimental group (8 nodes per group). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing twice 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 'Irga-Safe Plus' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a 3-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5% trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, 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 S.l.
- Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
other: alpha-hexylcinnamaldehyde in acetone:olive oil (4:1, v/v)
The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:
The validation- / positive control study was performed with alpha-hexylcinnamaldehyde in acetone:olive oil (4:1, v/v) using CBA/Ca mice (RCC Study Number A89831) from 09-AUG-2006 to 23-AUG-2006. The reliability check with alpha-hexylcinnamaldehyde indicated that the local lymph node assay as performed at RCC is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: - Group 2 (1%): 0.7 - Group 3 (2.5%): 1.4 - Group 4 (5%): 1.6 A dose-response relationship was observed. Calculation of the EC 3 value was not performed because no test concentrations produced a S.I. of 3 or higher.
other: disintegrations per minute (DPM)
Remarks on result:
other: - Control: 2229 - Group 2 (1%): 1573 - Group 3 (2.5%): 3063 - Group 4 (5%): 3481

Any other information on results incl. tables

Test item concentration % (w/v)   Measurement
Calculation Result
dpm - BGa) number of lymph nodes dpm per lymph nodeb) SI
--- BG I 40 --- --- --- ---
--- BG II 40 --- --- --- ---
--- CG 1 2229 2189 8 274 ---
1 TG 2 1573 1533 8 192 0.7
2.5 TG 3 3063 3023 8 378 1.4
5 TG 4 3481 3441 8 430 1.6
BG = Background (1 ml 5 % trichloroacetic acid) in duplicate
CG = Control Group
TG = Test Item Group
SI = Stimulation Index
a)The mean BG was calculated from the figures BG I and BG II values.
b)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

No deaths occurred during the study period. No clinical signs on the ears of the animals and no systemic findings were observed during the study period. The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information

Categories Display