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Diss Factsheets

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 study according to current guidelines

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)
Harlan Cytotest Cell Research GmbH
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Details on test material:
- Name of test material (as cited in study report): 2-Methyl-4,5,6,7,8,9,10,11,12, 13-decahydro-1 H-cyclopentacyclododecen
- Physical state: Liquid/ brown, turbid
- Analytical purity: 62.5 area-% (not corrected for the water content.)
- Lot/batch No.: Methyl-Cyclopentadien-Cyclododecan (Austrag Gasphase SVA-Fabrik 02-18.10.2012)
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: CBA/CaOlaHsd; Harlan Laboratories B.V., The Netherlands.
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 18.3g - 21.7g
- Housing: group
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: least 5 days

- Temperature (°C): 22+/-2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

methyl ethyl ketone
1%, 2% and 5%
No. of animals per dose:
Details on study design:
A solubility experiment was performed and the highest test item concentration, which could be technically used, was 100% of the undiluted test item. To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100%. This pre-experiment was cancelled and the animals euthanized approximately 4 hours after the first application, due to strong excitement observed in both animals.
Therefore, a second pre-test was performed and two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5 and 10% 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. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) 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.
On day 3, the animal treated with 10% test item concentration showed an erythema of the ear skin (score 1) and high activity, excessive scratching and cleaning behaviour even 3 hours after the third application. Increase in ear weight for the animal treated with 5% and 10% was 12.4 and 23.6%, respectively compared to historical vehicle values. The animal treated with 5% test item concentration did not show any signs of local skin irritation or systemic toxicity.
Thus, the test item in the main study was assayed at 1, 2 and 5%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

- 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.
In addition, an index was calculated for the lymph node weight and – cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle treated group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1. However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.

The test item preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 1, 2 and 5% in MEK. 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 (control animals).
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 the test item groups and negative control group. A One-Way-Analysis-of-Variance was used as a 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 was used for detection of possible outliers. However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: 1% TS group: 4.42 2% TS group: 5.83 5% TS group: 15.71
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle control: 822.9 (+/- 128.9 SD) 1% TS group: 3638.9 (+/- 628.9 SD) 2% TS group: 4800.7 (+/- 1054.9 SD) 5% TS group: 12929.9 (+/- 3814.6 SD)

Any other information on results incl. tables

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No signs of systemic toxicity 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

A statistically significant and biologically relevant increase in lymph node weights and – cell counts was observed in all test item treated groups in comparison to the vehicle control group. The indices determined for the lymph node cell count was exceeded in all dose groups compared to the cut-off value of 1.55.

Index values (test group vs control group) LN weights

1% TS group: 1.52

2% TS group: 1.33

5% TS group: 2.37

Index values (test group vs control group) LN cell count

1% TS group: 1.74

2% TS group: 1.67

5% TS group: 3.1

Ear Weights

A statistically significant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. The index determined for the group treated with a test item concentration of 5% slightly exceeded the cut-off value of 1.1. However, this was considered to be not biologically relevant, as the mean value of the groups was only slightly above the range of historical vehicle control data for the ear weight and the observed increase did not exceed the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429.

Index values (test group vs control group)

1% TS group: 1.03

2% TS group: 1.04

5% TS group: 1.14

Applicant's summary and conclusion

Interpretation of results:
Migrated information
The present data on dermal sensitization fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a classification with R43 and "Skin sensitisation" (Category 1A) is warranted.