Methoxycyclododecane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-31 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

(adopted July 2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

29 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Federal Office for the Environment (FOEN), Federal Office of Public Health (FOPH) and Swiss Agency for Therapeutic Products (Swissmedic)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methoxycyclododecane

EC Number:

221-053-8

EC Name:

Methoxycyclododecane

Cas Number:

2986-54-1

Molecular formula:

C13H26O

IUPAC Name:

methoxycyclododecane

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Venray, The Netherlands
- Age at study initiation: 9 weeks at the beginning of treatment
- Weight at study initiation: 19.2 - 22.2 g at the beginning of treatment
- Housing: Group housing in Makrolon Type-II cages with standard softwood bedding
- Diet: Pelleted standard Harlan Teklad 2914C rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst, Switzerland), ad libitum
- Water: Community tap water from Itingen, Switzerland, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10, 25 and 30%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Irritation: A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation. Two test substance concentrations were tested; a 75% and 100% concentration. At the highest concentration (undiluted item) the animals showed signs of significant irritation. Increase in ear thickness was 40% upon the third measurement. At both test item concentrations, mild to moderate erythema (erythema scoring grade 2) was observed on the ears of the animals after application.
Therefore, it was decided by the sponsor to lower the dose levels for the main test and assay the test substance at 10%, 25% and 50% (w/v) in acetone/olive oil 4/1 v/v.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by beta-scintillation
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures. The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation.

TREATMENT PREPARATION AND ADMINISTRATION:
The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance in each test group, once daily for three consecutive days. The control animals were treated the same as the experimental animals, except that the vehicle was administered instead of the test substance. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured in a β-scintillation counter.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique was assessed by the use of Alpha-hexyl cinnamic aldehyde. The validation- / positive control study was performed as a stand alone study at intervals not longer than 6 months. In the recent representative positive control validation study (D19153) S.I. values of 4.8, 5.8 and 24.5 were determined with the test item dissolved in acetoen/olive oil (4:1, v/v) at concentrations of 5%, 10% and 25% (w/v), respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 10, 25 and 50% were 1.6, 2.6 and 3.7 respectively. An EC3 value of 34.1% was derived (conctrations of 25 and 50% were used for calculation of the EC3 value).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 2701, 4370 and 6169 DPM respectively. The mean DPM/animal value for the vehicle control group was 1684.

Any other information on results incl. tables

No deaths occurred during the study period. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period. The body weights of the animals were within the range commonly recorded for animals of the strain and age.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the results in the LLNA the test material shows a sensitising potential when tested up to the concentration of 50% (w/v) in acetone/olive oil 4/1 v/v. The EC3 value calculated was 34.1%.