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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-07-06 to 2011-07-27
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(adopted 22 July 2010)
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(EC) No.440/2008, dated 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:
Test material form:
other: colourless liquid

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst, Netherlands
- Age at study initiation: Pre test: 10-11 weeks; Main study: 9-10 weeks
- Weight at study initiation: 19.1-24.6 g
- Housing: Group in cages Makrolon Type II /III, with wire mesh top
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days

- Temperature (°C): 22 +/- 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
2.5, 5, 10 %
No. of animals per dose:
5 animals/doses
Details on study design:
- Lymph node proliferation response: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.).

- Name of test method:LLNA
- 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.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item. Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline(PBS) containing 20.1 µCi of 3HTdR (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 by intraperitoneal injection of Pentobarbital-Natrium. The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). After preparing of single cell suspensions of pooled lymph node cells the level of 3HTdR incorporation was then measured on a -scintillation counter. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: Prior to the first application and prior to sacrifice. In the main experiment: Prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.
Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Lymph node cell count: The lymph node cell count were determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a Cell Counter.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.
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 DPM values (group mean DPM ± standard deviation). For all statistical calculations SigmaStat for Windows (Version 2.0) was used.
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.

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitisation properties in CBA/CaOlaHsd mice. 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 May 2011.

Stimulation Index: 1.00, 1.30, 2.82, 9.03 respectively to the concentrations:0, 5, 10, 25 %
The Estimated concentration for a S.I. of 3 (EC3) resulted in 10.4 % (w/v). EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: Stimulation Indices relative to the mean of the control group (Group 1) 0 %: 1 2.5 %: 1.7- 4.1 mean 2.71 5 %: 1.0- 2.1 mean 1.52 10 %: 2.0- 8.8 mean 4.49 The estimated concentration for a S.I. of 3. (EC3) resulted 7.5 %.
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
DPM mean value (per animal) corrected for mean background value (1 mL 5% trichloroacetic acid) 0 %: 172- 458 mean 266.9 2.5 %: 456- 1093 mean 723.3 5 %: 257- 563 mean 404.5 10 %: 522- 2340 mean 1197.1 The estimated concentration for a S.I. of 3. (EC3) resulted 7.5 %.

Any other information on results incl. tables

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Body Weights: 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: The measured lymph node weights and cell counts of all animals treated were recorded after sacrifice. In the high dose group, a statistically significant and biologically relevant increase in lymph node weight was observed in comparison to the vehicle control group (p < 0.05). The lymph node cell count showed no statistically significant increase in any test item treated group in comparison to the vehicle control group. However, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in this group and was therefore considered to be biologically relevant (index of 1.6).

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 low and the high dose group in comparison to the vehicle control group (p<0.05). However, the cutoff-value for a positive response regarding the ear weight index of 1.1 reported for BALB/c mice was not exceeded in any dose group. Still, the mentioned cutoff-value has been determined using a different strain of mice and can thus not be implicitly adopted.

Applicant's summary and conclusion

Interpretation of results:
Migrated information