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Diss Factsheets

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:
2011-10-26 to 2011-11-16
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline 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)
according to guideline
EU Method B.42 (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:
Test material form:
other: liquid
Details on test material:
Identity: 2-Methylbutan-2-ol

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: pre-test: 10-11 weeks (beginning of treatment)
main study: 8-9 weeks (beginning of treatment)
- Weight at study initiation: 16.2 - 20.8 g
- Housing: Group
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days

- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45 – 65%
- Air changes (per hr): About 10 / hour
- 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)
25, 50, 100 %
No. of animals per dose:
5 females per dose
Details on study design:
RANGE FINDING TESTS: a pre-test was performed in two animals .
- Treatment: topical application to the dorsal surface of each ear with test item concentrations of 50% (w/w) and 100% once daily each on three consecutive days.
- Systemic toxicity: At the tested concentrations the animals did not show any signs of systemic toxicity.
- Body weight: The animal treated with the undiluted test item showed loss of body weight of – 5.4%. Because no signs of systemic toxicity were observed in this animal, this was considered to be incidental.
- Irritation: On day 2 and day 3, the animal treated with the undiluted test item showed an erythema of the ear skin (Score 1). Other signs of irritation were not observed.


- Criteria used to consider a positive response:
a.) 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.
b.) 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 concentrations of 25, 50 (w/w), and 100% in acetone:olive oil (4+1 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface 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 20.2 µCi of 3HTdR (equivalent to approximately 80.9 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). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze. 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 (1 mL) and 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 beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). The obtained DPM values were used to calculate stimulation indices (SI).

After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. 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 (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
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). However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: 25 % 2-Methylbutan-2-ol: 1.6 * 50 % 2-Methylbutan-2-ol: 0.95 100 % 2-Methylbutan-2-ol: 1.13 * statistically significant increase vs. control group (p<0.05) The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Although a statistically significant increase in DPM value was observed in the low dose group in comparison to the vehicle control group, this was not considered as biologically relevant as the S.I. determined for this concentration did not exceed the threshold value of 3. Mean DPM - Background (1 mL 5% trichloroacetic acid): - Vehicle (acetone:olive oil (4+1 v/v)): 1127.8 +/- 321.4 - 25 % 2-Methylbutan-2-ol: 1800 +/- 383.5 - 50 % 2-Methylbutan-2-ol: 1075.4 +/- 281.3 - 100 % 2-Methylbutan-2-ol: 1274.6 +/- 263.4

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 after 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. A statistically significant or biologically relevant increase in lymph node weights and lymph node cell count was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not exceed this threshold.

Ear Weights

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response. This threshold was not exceeded in any test item treated group.

Applicant's summary and conclusion

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