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Skin sensitisation

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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: Guideline Study OECD 429 and GLP compliance

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)
GLP compliance:
yes (incl. QA statement)
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:
Details on test material:
- Name of test substance: N-(2-Hydroxyethyl)-ethylendiamin 99 %
- Test substance No.: 05/0697-1
- Batch No.: 11001DB065
- Homogeneity: The test substance was homogeneous by visual inspection.
- Stability: The stability under storage conditions was confirmed by reanalysis

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Origin: Harlan Winkelmann GmbH, Gartenstr. 27, 33178 Borchen, Germany
- Sex: Female
- Identification of the animals: The single housed animals were identified by cage cards
- Age at the beginning of the study: ca. 7 weeks
- Body weight range at day 0: 17.1 g – 20.7 g
- Reasons for the selection: The test method was developed in mice.

Housing conditions:
- Air conditions: The animals were housed in fully air-conditioned rooms in which a central air-conditioning system ensured a temperature in the range of 20 - 24 °C and a relative humidity in the range of 30 - 70 %.

- Illumination period: 12 h light (6.00 a.m. - 6.00 p.m.) , 12 h darkness (6.00 p.m. - 6.00 a.m.)
- Type of cage: Makrolon type I
- No. of animals per cage: 1
- Type of diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Watering: Tap water ad libitum
- Bedding: Granulat Typ ¾ (staubfrei); SSNIFF
- Acclimatization period: 8 days before the first test substance application

Study design: in vivo (LLNA)

other: acetone
1) 3 %
2) 10 %
3) 30 %
4) vehicle acetone
No. of animals per dose:
Details on study design:
The study comprised three treatment groups and a vehicle control group. Each group consisted of 6 mice. A check for dead or moribund animals was made twice each workday and once on Saturdays, Sundays and on public holidays.
No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data.

- Body weights of the individual animals were determined on study day 0 prior to the first application and on day 5 prior to the sacrifice of the animals
- Test substance application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 μL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously (i.v.) with 20 μCi of 3H-thymidine in 250 μl of sterile saline into a tail vein.

- The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.

- Immediately after the death of each animal ear thickness was measured with a suitable gauge (Oditest®-device (measurement steps 0.01 mm), Kroeplin, Germany). Thereafter a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.

- Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of cell count, 3H-thymidine incorporation, lymph node weight, ear weight and ear thickness measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group. Further statistical analyses ( Cell counts, 3H-thymidine incorporation, lymph node weight, ear weights as well as ear thickness) were performed with the WILCOXON-Test.

Results and discussion

Positive control results:
The Murine Local Lymph Node Assay is able to show the skin sensitizing effect of Alpha-Hexylcinnamaldehyde, techn. 85 % under the test conditions chosen. The threshold concentration for sensitization induction was between 5 % and 10 % under the test conditions chosen. The EC 1.5 for cell count and the EC 3 for 3H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 6.9 % and 10.5 % , respectively.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: control (vehicle) 1.0, 3 % AEEA 2.0, 10 % AEEA 1.72, 30 % AEEA 6.6 EC 3: 15.2 %
other: disintegrations per minute (DPM)
Remarks on result:
other: Means: control (vehicle) 444.9, 3 % AEEA 891.1, 10 % AEEA 766.1, 30 % AEEA 2938.3

Any other information on results incl. tables

- Cell counts, 3H-thymidine incorporation and lymph node weights

When applied as 30 % preparation in acetone, the test substance induced a statistically significant and biologically relevant (increase to 1.5 fold or above of control value = stimulation index (SI) 1.5) increase in cellularity of the auricular lymph nodes. Concomitantly, the increase of 3H-thymidine incorporation into the cells was statistically significant and biologically relevant (increase above the cut off stimulation index of 3). The 10 % and 3 % test substance preparation caused small but statistically significant increases in cellularity of the auricular lymph nodes and 3H-thymidine incorporation into the lymph node cells, which failed to reach the cut off stimulation indices and thus lie below the threshold of immunologic relevance. The lymph node weights were statistically significantly increased in all substance treated groups.

- Ear weights and ear thickness

Statistically significant increases in ear weights and ear thickness were observed in mice treated with the 30 % test substance preparation, which was accompanied by incrustation of the ear skin observed before the third application and on the day of lymph node removal. The 10% test substance preparation caused a statistically significant increase in ear weights, but not in ear thickness. The magnitude of ear skin irritation observed in the test group treated with a 30 % preparation might be considered relevant for evaluating the sensitizing potential of the test substance. As the stimulation indices at this concentration, especially that for cellularity, are well beyond the cut off criteria, an immunologic background of the lymph node response cannot be excluded.

Applicant's summary and conclusion

Interpretation of results:
Migrated information
Executive summary:

In a dermal sensitisation study with AEEA (BASF AG, Experimental Toxicology and Ecology, 2006) groups of 6 female CBA/Ca mice each were treated with 3 %, 10 % and 30 % w/w preparations of the test substance in acetone or with the vehicle alone using the Local Lymph Node Assay.AEEA shows a skin sensitizing effect under the test conditions chosen. The threshold concentration for sensitization induction was between 10 % and 30 %. The EC 3 for ³H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 15.2 %.