Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Justification for type of information:
see chapter 13 read across justification

Data source

Referenceopen allclose all

Reference Type:
study report
Report Date:
Reference Type:
other: Amendment
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
according to
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
according to
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Bioassay Labor für biologische Analytik GmbH, INF 515, 69120 Heidelberg, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Details on test material:
- Name of test material (as cited in study report): Reaction mass of 2,2´-[(4-methylphenyl)imino]bisethanol and Ethanol 2-[[2-(2(hydroxyethoxy)ethyl](4-methylphenyl)amino]-
- Batch No.: 75849288Q0
- Purity: 99.4%
- Physical state, colour: Liquid/viscous/yellowish, clear
- Storage conditions: Room temperature, under N2
- Expiration Date: September 09, 2014

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 18.2 - 21.0 g
- Housing: single in Markrolon cages, type II; H 15005-29 bedding; Ssniff.Spezialitäten GmbH (Experimental AnimalDiets Inc., 59494 Soest, Germany)
- Diet: STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing

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

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
25, 50, 100% (w/w)
No. of animals per dose:
Details on study design:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 442B. The highest test item concentration, which could be technically used, was 100%.
- Irritation: To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in six animals (2 animals per test item concentration and vehicle, each). Six mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% (w/w) and with the vehicle once daily each on three consecutive days. At the tested concentrations of 50 and 100% the animals showed no relevant signs of local irritation as confirmed by ear weight measurements and ear swelling measurements. Very slight erythema and test item residues (colorless) were noted in all animals; in addition incrustations were observed in one animal prior sacrifice only. Therefore, the highest concentration tested in the main study was 100%

- Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 25, 50, and 100% (w/w) in the vehicle. The application volume 25 μL/ear/day was spread over the entire dorsal surface (Ø ~8 mm) of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was treated with 25% α-hexyl cinnamaldehyde dissolved in acetone: olive oil (4:1 v/v).
- Administration of BrdU: BrdU was purchased from Roche Diagnostics GmbH. For injection it was dissolved in DPBS (10 mg/mL) before administration and stored in a refrigerator until use. Four days after the first topical application (day 5) 0.5 mL of BrdU solution (5 mg/per mouse/injection) were intraperitoneally injected.
- Determination of Incorporated BrdU: Approximately 24 hours after treatment with BrdU the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a 70 μm nylon mesh. The lymph node cells were resuspended in 6 mL DPBS. After cell count with a cell counter (Casy Cell Counter, Innovatis), cell suspensions of 100,000 cells / mL were adjusted. The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit as follows (Roche Applied Science. Mannheim): 100 μL of the lymph node cell suspension (100,000 cells /mL) were added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the lymph node cells with FixDenat (included in Cell Proliferation Elisa Kit), anti BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution TMB (included in Cell Proliferation Elisa Kit) was then added and allowed to produce chromogen. After stopping the substrate reaction the absorbance was measured at 450 nm with a reference wavelength of 690 nm (Absorbance-Reader SUNRISE,Tecan).
- Determination of Lymph Node Weight and Cell Count: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance (XS 204, Mettler Toledo). Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned above was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (Casy Cell Counter, Innovatis). The values obtained were taken down manually.
- Determination of Ear Weights: After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance (XS 204, Mettler Toledo).

The test item preparations were produced on a weight per weight (w/w) basis. The test item was emulsified in the vehicle AOO. The positive control preparation was produced on a weight per weight (w/w) basis shortly before application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer the positive control was soluble in the vehicle AOO.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the BrdU values. For the calculations excel (Version 2010) was applied. No EC1.6 value could be calculated, because all S.I.s obtained were above the threshold of 1.6. A statistical analysis was conducted on the BrdU values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations Statistica (Version 10) was used. A Mann-Whitney-U test for non-parametric comparison was applied as statistical test. Statistical significance was set at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
A stimulation indice of 2.9 was determined for 25% alfa-hexyl cinnamaldehyde in acetone: olive oil (4:1 v/v) in the current experiment. Stimulation Indices of 2.6, 5.0, 5.4, 1.9 and 3.4 were determined in 5 previous consecutive positive control experiments, with 25% alfa-hexyl cinnamaldehyde in acetone: olive oil (4:1 v/v).

In vivo (LLNA)

Remarks on result:
other: see Remark
In this study Stimulation Indices (S.I.) of 2.8, 3.4 and 4.1 were determined with the test item at concentrations of 25, 50 and 100% (w/w) in AOO, respectively. An EC1.6 value could not be calculated, because the S.I. values in every dose group exceeded the value of 1.6. A clear dose response was observed.

Any other information on results incl. tables

No deaths occurred during the study period. The animals did not show any signs of systemic toxicity. Very slight erythema was observed in the 100% dose group during the course of the study, while scaling was noted in the 50% dose group prior sacrifice only. The positive control group showed very slight erythema during the course of the study in addition to scaling prior sacrifice. The body weight of the animals recorded prior to the first application and prior to sacrifice were within the range commonly recorded for animals of this strain and age. 

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weights and - cell counts was observed in the 25%, 50% and 100% dose group and in the positive control in comparison to the vehicle control group. 

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was not observed in the test item groups. In the positive control ear weight measurement reached statistical significance without biological relevance in comparison to the vehicle control group. According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was neither exceeded in any test item treated group nor in the positive control.

Applicant's summary and conclusion

Interpretation of results:
Migrated information