[1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: 3rd Jun 2011
Experimental Completion Date: 15 Jun 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Exceptions: 1) The test article dosing formulations were not analyzed for concentration, homogeneity, or stability. The impact on this study is unknown since the actual dose of the test article dosing formulation cannot be confirmed.

2) Stability data for the test article was not provided. However, there was no impact on the study since an expiration data was provided on the Certificate of Analysis and the study was conducted before the expiration date.

3) It is unknown if the test article characterization was done under GLP/GMP. However, a Certificate of Analysis was provided. Therefore, there was no impact on the study

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

impurity 1

Reference substance name:

[1S-(1α,2α,3aβ,4α,8aβ,9R*)]-decahydro-1,5,5,8a-tetramethyl-1,2,4-methenoazulene

EC Number:

214-504-5

EC Name:

[1S-(1α,2α,3aβ,4α,8aβ,9R*)]-decahydro-1,5,5,8a-tetramethyl-1,2,4-methenoazulene

Cas Number:

1137-12-8

Molecular formula:

C15H24

IUPAC Name:

(1S,2S)-1,2,6,6-Tetramethyltetracyclo[8.1.0.02.8.07.11]undecane

impurity 2

Reference substance name:

(+)-Sativene

Cas Number:

3650-28-0

Molecular formula:

C15H24

IUPAC Name:

(+)-Sativene

impurity 3

Reference substance name:

Caryophyllene

EC Number:

201-746-1

EC Name:

Caryophyllene

Cas Number:

87-44-5

Molecular formula:

C15H24

IUPAC Name:

(1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene

Constituent 1

Reference substance name:

[1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene

EC Number:

207-491-2

EC Name:

[1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene

Cas Number:

475-20-7

Molecular formula:

C15H24

IUPAC Name:

4,8,8-trimethyl-9-methylenedecahydro-1,4-methanoazulene

Test material form:

liquid

Specific details on test material used for the study:

Identification: Longifolene
Supplier: International Flavors and Fragrances
Batch No.: SM10096287
Expiration Date: Jun 2013
Physical Description: Clear colorless liquid
Storage Conditions: 19-23°C, Room temperature
Composition/Purity Stability: A Certificate of Analysis was provided and is presented in Appendix II.Stability data were not provided.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Age Range: 8-12 weeks at start of dosing; records of dates of birth for animals used in this study are retained in the Calvert archives.
Body Weight Range: 20-26 grams at the outset (Day 1) of the study
Animal Source: Jackson Laboratories, Bar Harbor, ME 04609
Experimental History: Purpose-bred and experimentally naïve at the outset of the study
Identification: Tail marked with an indelible marker
Housing: Animals were group-housed 5 per cage upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". Animals were also group-housed during the study (5 per cage). The room in which the animals were kept was documented in the study records. No other species were kept in the same room.
Lighting: 12 hours light/12 hours dark
Room Temperature: 24 to 28°C
Relative Humidity: 16 to 69%
Food: Animals had access to Harlan Teklad Certified Rodent Chow 2016C ad libitum. The lot number(s) and specifications of each lot used are archived at Calvert. No contaminants were known to be present in the certified diet at levels that would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer, records of which are maintained in the Calvert archives.
Water: Tap water was available ad libitum, via water bottles. The water is routinely analyzed for contaminants as per Calvert SOP's. No contaminants were known to be present in the water at levels that would be expected to interfere with the results of this study. Results of the water analysis are maintained in the Calvert archives.
Acclimation: Study animals were acclimated to their housing for six days prior to their first day of dosing.
All animals used in this study were assessed as to their general health.


The mouse is the standard species used in the local lymph node assay (LLNA), which has been developed as an alternative to the Guinea Pig sensitization tests. The LLNA is a refinement in terms of reducing or eliminating distress in the animals compared to the Guinea Pig tests. The number used is the minimum number recommended. (NIH Publication No. 99-4494).

Study design: in vivo (LLNA)

Vehicle:

other: 3:1 Diethyl Phthalate:Ethanol (3:1 DEP:EtOH)

Concentration:

On each day of dosing, the test article was prepared at 2.5%, 5%, 10%, 25% or 50% (v/v) in volumetric flasks by mixing the appropriate amount of test article in the vehicle. The test article dosing solutions were clear, colorless liquids.
A volume of 25 µI/ear was applied using a micro pipette.

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Irritation: Irritation was scored and recorded using the Draize scoring system. Scoring was performed prior to dosing on Days 1-3.
- Systemic toxicity: the doses have been selected so that the highest concentration maximizes
exposure while avoiding systemic toxicity and excessive local irritation.
- Erythema scores: as follows
Erythema and Eschar Formation (Most severely affected area graded):
No erythema: 0, Very slight erythema (barely perceptible): 1, Well-defined erythema: 2, Moderate to severe erythema: 3, Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
Animals will be selected by body weight and apparent good health. Groups of 5 CBNJ female mice will then be randomly assigned to study groups. The criterion for a positive response is that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of 5 CBNJ female mice were treated on the dorsal surface of both ears once per day for 3 days with 2.5%, 5%, 10%, 25% or 50% (v/v) of Longifolene, or with the vehicle (3:1 Diethyl Phthalate:Ethanol [3:1 DEP:EtOH]). On Day 6, the mice were injected, i.v., with 20 µCi of 3H-thymidine in sterile saline. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells from individual mice (un-pooled) were precipitated with 5% trichloroacetic acid (TCA) and the pellets counted in a ß-scintillation counter to determine incorporation of the 3H-thymidine

Positive control substance(s):

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

Statistics:

The mean DPM for each group was determined. Increases in 3H-thymidine incorporation relative to the vehicle­ treated control was derived for each group and recorded as stimulation indices (SI). The criterion for a positive response was that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.
The evaluation of the equality of means for body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.
Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM and body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.

Since the data indicated that the test article was positive, the EC3 was calculated using the formula:

EC3 = c+[(3-d)/(b-d))(a-c)

where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a,b) and (c,d) respectively.

Results and discussion

Positive control results:

The positive control gave the expected results.
Mean body weights at Day 1 and Day 6 and mean changes in body weights were evaluated. There were no statistically significant different in the mean body weights on Day 1 or Day 6 or in the mean body weight changes of the mice treated with Longifolene when compared to the vehicle control treated group. Therefore, administration of the test article did not appear to exert any overt toxicity.

In vivo (LLNA)

Results

Key result

Parameter:

EC3

Value:

9.4

Test group / Remarks:

treatment with Longifolene at concentrations of 10%

Any other information on results incl. tables

There was no mortality and all animals appeared normal throughout the study.

No erythema or edema was noted in any of the mice in the vehicle group or in those dosed with the test article at 2.5%, 5%, 10%, 25% or 50% (v/v). The ears of the mice treated with the vehicle appeared wet on Days 2 and 3. The ears of the mice appeared wet on Day 3 in the groups treated with the test article at 25% and on Days 2 through 4 in the group treated with test article at 50%. There were no other findings.

Mean body weights at Day 1 and Day 6 and mean changes in body weights were evaluated. There were no statistically significant different in the mean body weights on Day 1 or Day 6 or in the mean body weight changes of the mice treated with Longifolene when compared to the vehicle control treated group. Therefore, administration of the test article did not appear to exert any overt toxicity.

At termination, the lymph nodes from the mice in the vehicle group and in the groups treated with test article at 2.5%, 5%, 10%, 25% and 50% (v/v) were all normal in size and appearance.

Exposure to Longifolene at concentrations of 2.5%, 5%, 10%, 25% or 50% (v/v)

resulted in stimulation indices of 1.1, 1.6, 3.2, 3.7, and 5.4, respectively. In addition, the responses with 10%, 25% and 50% of the test article were statistically significant (p<0.001) when the log DPM was compared to the vehicle group. Since the data indicated a positive response the EC3 was calculated and determined to be 9.4%.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Remarks:

Skin sensitisation category 1B has been assigned according to the CLP regulation. Classification criteria as the 1B according to CLP is an EC3 value > 2 %. The EC3 value was calculated to be 9.4% and therefore it is classified as category 1B.

Conclusions:

A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index ≥ 3.0 is regarded as a positive response. Therefore, based on the criteria of this study, treatment with Longifolene at concentrations of 10% and above resulted in a stimulation indices of greater than 3.0 and hence it is considered to have skin sensitizing activity. The EC3 was calculated to be 9.4%.
Skin sensitisation category 1B has been assigned according to the CLP regulation.
Classification criteria as the 1B according to CLP is an EC3 value > 2 %.
The EC3 value was calculated to be 9.4% and therefore it is classified as category 1B.

Executive summary:

The purpose of this study was to determine if the test article would induce a hypersensitivity response in mice as measured by the proliferation of lymphocytes in the draining lymph nodes.