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
Study period:
2012-01-29 to 2012-03-06
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline compliant study

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 22, 2010
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 30, 2008
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:

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 1st pre-test: 9-10 weeks
2nd pre-test: 10-11 weeks
main study: 9-10 weeks
- Housing: 5 animals per cage, Makrolon Type IIII with wire mesh top
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days prior to treatment

- Temperature (°C): 22 ± 2
- Humidity (%): 32 – 65% (acclimation period)
30 – 65% (pre-test)
45 – 65% (main study)
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
0 (control) 5, 10, 25% test item in vehicle
No. of animals per dose:
pre-test: 4 females (2 for each pre-test)
main-test: 20 females (5 females per dose group)
Details on study design:
- Compound solubility: The highest test item concentration, which could be technically used was the undiluted test item (100%). Test item solution at different concentrations was prepared using acetone:olive oil (4+1 v/v) as vehicle.
- Irritation: Two groups of two mice each were treated by topical application to the dorsal surface of each ear with test item concentrations of 50% (w/w) and 100% (undiluted test item) once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily and eventual signs of local irritation were scored. Prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined. Additionally, the ears of all animals were punched after sacrifice (day 6) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation.
At the tested concentrations the animals did not show any signs of systemic toxicity. Signs of ear skin irritation (erythema) were noted at all test concentrations. For the test concentrations of 100% and 50% the measured ear weight showed an increase that exceeded the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429.
Due to these findings, the test item in the main study was assayed at 5, 10, and 25% (w/w).

- Methodology: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in acetone:olive oil (4+1 v/v). 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. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 20.1 µCi of 3H-methyl thymidine (equivalent to approximately 80.4 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised. 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. After washing two times with phosphate buffered saline the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid, transferred to plastic 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 measured in two aliquots of 5 % trichloroacetic acid.
After excision, the lymph nodes were pooled per animal and weighed. Furthermore, the lymph node cell count was determined for each animal.
After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed (pooled per animal).

- Criteria used to consider a positive response: 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 concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
A statistical analysis was conducted on the DPM 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. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
The periodic positive control experiment was performed with α-hexyl cinnamaldehyde dissolved in acetone:olive oil (4:1 v/v) using CBA/CaOlaHsd mice in December 2011 (periodic positive control experiments are performed at intervals of not longer than 6 months) and revealed the expected result (EC3 of 14.4%) demonstrating the sensitivity and reliability of the experimental technique employed.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: 1.00 (vehicle group), 0.76 (5% 2-morpholinoethanol), 1.34 (10% 2-morpholinoethanol) and 2.52 (25% 2-morpholinoethanol). The EC3 value could not be calculated, since all S.I.s were below the threshold value of 3.
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Mean DPM values per animal: 399.5 (control group, just vehicle applied), 301.9 (5% 2-morpholinoethanol), 533.9 (10% 2-morpholinoethanol) and 1005.1 (25% 2-morpholinoethanol). The DPM value obtained for one animal of the control was identified as outlier and was excluded from calculation of the Mean and S.I. of this group.

Any other information on results incl. tables

Statistical evaluation of the dpm values

With or without inclusion of the outlier in the control group, a statistically significant increase in DPM value was not observed in any test item treated group in comparison to the vehicle control group.

Mortality, Clinical Signs and Body Weigths

No deaths occurred during the study period. Regarding clinical signs on day 2 to 6, the animals belonging to the high dose group showed an erythema of the ear skin (Score 1 on day 2 and 6, Score 2 on day 3 to 5). The animals of the mid dose group also showed an erythema of the ear skin (Score 1 on day 3, Score 2 on day 4, and Score 1 on day 5 and 6). In the animals of the low dose group, an erythema of the ear skin (Score 1) was observed from day 3 to day 6. The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Lymph Node Weights and Cell Counts

A statistically significant increase in lymph node cell count was observed in the highest dose group in comparison to the vehicle control group (p≤0.05). For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. According to this criterion, the lymph node cell count index determined for the high dose group (1.55) indicates a borderline response.

No statistically significant increase in lymph node weights was observed in any test item treated group in comparison to the vehicle control group.

Ear Weights

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p≤0.05). The cutoff-value of 1.1 of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was exceeded in this dose group (index of 1.2).

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU