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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Cas Number:
28973-99-1
Molecular formula:
C15H24
IUPAC Name:
(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Constituent 2
Chemical structure
Reference substance name:
(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene
Cas Number:
25532-79-0
Molecular formula:
C15H24
IUPAC Name:
(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene
Constituent 3
Chemical structure
Reference substance name:
(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)
Cas Number:
29837-07-8
Molecular formula:
C15H24
IUPAC Name:
(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)
Constituent 4
Chemical structure
Reference substance name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
EC Number:
242-582-0
EC Name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Cas Number:
18794-84-8
Molecular formula:
C15H24
IUPAC Name:
7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Constituent 5
Chemical structure
Reference substance name:
(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Cas Number:
53585-13-0
Molecular formula:
C15H24
IUPAC Name:
(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Constituent 6
Chemical structure
Reference substance name:
(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Cas Number:
13062-00-5
Molecular formula:
C15H24
IUPAC Name:
(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Constituent 7
Chemical structure
Reference substance name:
1-methyl-4-(6-methylhepta-1,5-dien-2-yl)
Cas Number:
869843-05-0
Molecular formula:
C15H24
IUPAC Name:
1-methyl-4-(6-methylhepta-1,5-dien-2-yl)
Constituent 8
Chemical structure
Reference substance name:
(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Cas Number:
26560-14-5
Molecular formula:
C15H24
IUPAC Name:
(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Constituent 9
Chemical structure
Reference substance name:
(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Cas Number:
28973-97-9
Molecular formula:
C15H24
IUPAC Name:
(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Constituent 10
Chemical structure
Reference substance name:
1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene
Cas Number:
72345-84-7
Molecular formula:
C15H24
IUPAC Name:
1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene
Constituent 11
Chemical structure
Reference substance name:
2,6,10-trimethyldodeca-2,6,9,11-tetraene
EC Number:
207-948-6
EC Name:
2,6,10-trimethyldodeca-2,6,9,11-tetraene
Cas Number:
502-61-4
Molecular formula:
C15H24
IUPAC Name:
3,7,11-trimethyldodeca-1,3,6,10-tetraene
Constituent 12
Chemical structure
Reference substance name:
Likely sesquiterpene hydrocarbons
Cas Number:
n/a
Molecular formula:
C15H24
IUPAC Name:
Likely sesquiterpene hydrocarbons
Test material form:
liquid
Details on test material:
UVCB substance
Specific details on test material used for the study:
Batch number: 9000458190
Stable under storage conditions: in the original container in refrigerator (4 +/- 3 °C), away from direct sunlight

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
4 females per group, 4 test groups, 1 control (vehicle) group. Age between 7 and 12 weeks, body weights between 16.4g and 20.2g.
Identification by unique cage card and individual color code.
Randomization: randomly selected by computer algorithm at time of delivery.
Acclimatization under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Animals were kept in Standard Laboratory Conditions:
Air-conditioned with target ranges for romm temperature 17-23°C, relative humidity 30-70% and approximately 10-15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges may have occasionally occured, usually following room cleaning. These transiet variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the daytime light period.
Accomodation: In group of four in Makrolon type-3 cages with standard softwood bedding.
Diet: Pelleted standard Provimi Kliba 3433 mouse maintenance diet available ad libitum provided by Provimi Kliba AG, CH-4303 Kaiseraugst. Results of analysis for contaminants are archived at RCC lab.
Water: Community tap water from Itingen, availablead libitum. Results for bacteriological, chemical and contaminant analyses are archived at RCC lab.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Remarks:
ethanol: water, 7:3 (v/v)
Concentration:
0.1%, 1%, 10% in ethanol:water, 7:3 (v/v) and 100% undiluted.
No. of animals per dose:
4
Details on study design:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe with different test item concentrations. The application volume, 25ul, was spread over the entire dorsal surface of each ear lobe once daily for the three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine then 5 hours after this the mice were sacrificed.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
8.6
Parameter:
SI
Value:
0.8
Test group / Remarks:
Test item concentration 1% (w/v))
Parameter:
SI
Value:
3.4
Test group / Remarks:
Test item concentration 10% (w/v))
Parameter:
SI
Value:
0.4
Test group / Remarks:
Test item concentration 0.1% (w/v))
Cellular proliferation data / Observations:
No deaths occured during the study period.
No test item-related clinical signs were observed in all animals with the exception of the group 5 (100% undiluted). About one hour after the first application a moderate swell was observed at the dosing sites of both ears in all animals of Group 5 (100% undiluted). The swelling reduced to slight five days after the first dosing. Two days after the first local application a slight general erythema showed at the dosing sites of both ears all mice of the group 5 (100% undiluted). This lasted for days remained.
The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this train and age.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In this study stimulation indices of 0.4, 0.8, 3.4 and 13.5 were determined with the test item at concentrations of 0.1, 1, 10% (w/v) in ethanol: water, 7:3 (v/v) and 100% (undiluted).
A test item is regarded as a sensitizer in the LLNA if the exposure to at leasr 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.
Based on these criteria, the test item Bisabolene was found to be a non-sensitizer when tested up to 1% (w/v) in ethanol: water, 7:3 (v/v).
Bisabolene showed an allergenic potency when tested at concentrations of 10% (w/v) in ethanol: water, 7:3 (v/v) and 100% (undiluted).
An EC3 value of 8.6% (w/v) was derived.
Executive summary:

In order to study a possible allergenic potential of Bisabolene, four groups of four female mice were each treated with the test item at concentrations of 0.1%, 1%, 10% in ethanol:water, 7:3 (v/v) and 100% undiluted by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only ethanol:water, 7:3 (v/v). Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine. Approximatelly five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pool lymph nodes which were washed subsequently ans incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of thymidine measured in a beta-scintillation counter.

 

No test item-related clinical signs were observed in all animals with the exception of the group 5 (100% undiluted). About one hour after the first application a moderate swell was observed at the dosing sites of both ears in all animals of Group 5 (100% undiluted). The swelling reduced to slight five days after the first dosing. Two days after the first local application a slight general erythema showed at the dosing sites of both ears all mice of the group 5 (100% undiluted). This lasted for days remained.

All treated animals survived the scheduled study period.

 

A test item is regarded as a sensitizer in the LLNA if the 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.

The estimated concentration of test material required to produce a stimulation index of 3 is referred to as the EC3 value.

 

Based on these criteria, the test item Bisabolene was found to be a non-sensitizer when tested up to 1% (w/v) in ethanol: water, 7:3 (v/v).

Bisabolene showed an allergenic potency when tested at concentrations of 10% (w/v) in ethanol: water, 7:3 (v/v) and 100% (undiluted).

An EC3 value of 8.6% (w/v) was derived.

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