Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 8, 2010 - June 30, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study under GLP with full documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 39 - 81 % for few hours. This deviation to the study plan, however, does not affect the validity of the study.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 39 - 81 % for few hours. This deviation to the study plan, however, does not affect the validity of the study.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- The analysis was performed for the reference sample "Sample 2010" as defined in Section 1.4
- Name of test material (as cited in study report): reaction mass of 2,2’-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA)
- Analytical purity: 100 % mixture
- Lot/batch No.: Ref.Nr 10013591
- Expiration date of the lot/batch: March 2011
- Stability under test conditions: Not indicated by the sponsor
- Storage condition of test material: At +2 to +8 °C, light protected, under nitrogen

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: 18.4 - 23.6 g at the start of the main experiment
- Housing: Single caging. The animals were distributed into the test groups at random and identified by cage number.
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst / Netherlands)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 39-81%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: Not applicable
Vehicle:
other: Not applicable
Concentration / amount:
Not applicable
Challengeopen allclose all
Route:
other: Not applicable
Vehicle:
other: Not applicable
Concentration / amount:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Challenge controls:
Not applicable
Positive control substance(s):
not required
Remarks:
Not applicable

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 or 100%
No. of animals per dose:
4 females per group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was 100% of the undiluted test item.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 50 and 100% each on three consecutive days. In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
- Lymph node proliferation response: Not reported

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: (1) 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. (2) 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.


TREATMENT PREPARATION AND ADMINISTRATION:
Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50, and 100% (w/w) in acetone:olive oil (4:1). The application volume, 25 ul, was spread over the entire dorsal surface (diameter approximately 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Application was without occluded patch.

Endpoint to measure effect:
Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intraveinous injection, the mice were sacrificed and the draining auricular lymph nodes 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 proliferate capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables

Results and discussion

Positive control results:
Control group: 4549 DPM
5% group: 9263 DPM
10% group: 15495 DPM
25% group: 27949 DPM

Stimulation indexes were 2.04, 3.41 and 6.14 for the 5%, 10% and 25% groups respectively

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 25% group: 0.84 50% group: 1.14 100% group: 1.34
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control group: 3494 25% group: 2928 50% group: 3971 100% group: 4677

Any other information on results incl. tables

No deaths occurred during the study period.

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

The body weight of the animals, recorded prior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The sensitisation properties of Reaction mass of 2,2'-oxybisbutane and 2-methylpropan-2-ol and butan-2-ol and diisopropyl ether were tested with the LLNA method. No sensitisation effect were observed. Consequently, the test item is not sensitising.
Executive summary:

In the study the test item reaction mass of 2,2’-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA) dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential in this study under GLP. The guidelines OECD 429 and EU B.42 were followed.

For this purpose a local lymph node assay was performed using test item concentrations of 25, 50, and 100%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 0.84, 1.14, and 1.34 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4+1), respectively.

The test item reaction mass of 2,2’-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA) was not a skin sensitiser.