[(3,7-dimethyl-6-octenyl)oxy]acetaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 20, 2009 to September 10, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

[(3,7-dimethyl-6-octenyl)oxy]acetaldehyde

EC Number:

231-324-2

EC Name:

[(3,7-dimethyl-6-octenyl)oxy]acetaldehyde

Cas Number:

7492-67-3

Molecular formula:

C12H22O2

IUPAC Name:

2-[(3,7-dimethyloct-6-en-1-yl)oxy]acetaldehyde

Test material form:

liquid

Specific details on test material used for the study:

Identification: LLNA-384 (138650 Muguet Aid 50 PCT DEP BHT, Muguet Aldehyde 50% DEP)
Lot/Batch No.: 401893
Supplier: International Flavors & Fragrances (IFF-R&D)
Expiration Date: Aug 2010
Physical Description: Clear colorless liquid
Composition/Purity: A Certificate of Analysis was provided and is located in Appendix Ill

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Age range: 10 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: 19-25 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

Study design: in vivo (LLNA)

Vehicle:

other: Diethyl Phthalate (Dieth Phthalate Special Odorless, DEP)

Concentration:

The test article was received at 50% and therefore the 50% dose group was dosed as received. On each day of dosing, the test article was prepared at 2.5%, 5%, 10%, or 25% (v/v) in volumetric flasks by mixing the appropriate amount of test article in the vehicle.

No. of animals per dose:

Vehicle control: 5 animals
Dose of 2.5% (based on fragrance ingredient): 5 animals
Dose of 5% (based on fragrance ingredient): 5 animals
Dose of 10% (based on fragrance ingredient): 5 animals
Dose of 25% (based on fragrance ingredient): 5 animals
Dose of 50% (as received): 5 animals

Details on study design:

Dosing:
- Route: Typically on the dorsal surface of both ears
- Frequency: Once daily for 3 consecutive days (Days 1-3). The timing of dose administration remained consistent(± 2 hours) during the dosing phase.
- Procedure: A volume of 25 µI/ear was applied using a micro pipette.

Justification for Route and Dose Levels: The dermal route was selected as this is the route required for this model of hypersensitivity. In general, the doses were selected so that the highest concentration maximizes exposure while avoiding systemic toxicity and excessive local irritation. Doses were selected based on known reported uses of the material.

Method of performance: Mice were treated on the dorsal surface of both ears, once per day on Days 1, 2, and 3. Approximately 24±2 hours separated each application of test article. On Day 6, the mice were injected i.v. with 20 µCi of 3H -thymidine in 250 µI of sterile saline. Five hours rater the mice were euthanized by CO2 asphyxiation. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8°C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scinti[lation fluid. An additional 1 ml of TCA was used to rinse the tubel and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a B-scintilration counter.

Positive control substance(s):

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

Results and discussion

Positive control results:

HCA showed a SI of greater than 3.0 indicating a positive result and that the test was valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

28.3

Test group / Remarks:

Treatment with 50% of LLNA-384

Remarks on result:

other:

Remarks:

Mice treated with the test article at 25 and 50% also had ears that appeared wet on Days 5 and 6. There were no other findings. At termination, the lymph nodes from the mice in the vehicle group and the test article treated animals at 2.5%, 5%, 10%, 25% and 50% (v/v) were normal in size and appearance.

Results 2

Parameter:

SI

Value:

1.8

Test group / Remarks:

Group 2 - Dose: 2.5%

Results 3

Parameter:

SI

Value:

1.8

Test group / Remarks:

Group 3 - Dose: 5%

Results 4

Parameter:

SI

Value:

1.7

Test group / Remarks:

Group 4 - Dose: 10%

Results 5

Parameter:

SI

Value:

2.8

Test group / Remarks:

Group 5 - Dose: 25%

Results 6

Parameter:

SI

Value:

4.3

Test group / Remarks:

Group 6 - Dose: 50%

Cellular proliferation data / Observations:

Mortality: There was no mortality and all animals appeared normal throughout the study.
Clinical Observations: 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) (Table 1). Ears that appeared wet were observed in all mice treated with the vehicle and 2.5, 5, 10, 25 and 50% of the test article on Days 2-4. Mice treated with the test article at 25 and 50% also had ears that appeared wet on Days 5 and 6. There were no other findings.
Body Weights: The mean body weights and body weight changes of the mice treated with LLNA-384 on Days 1 and 6 showed no statistically significant differences (Table 2). Therefore, administration of the test article did not appear to exert overt toxicity.
Local Lymph Node Assay: At termination, the lymph nodes from the mice in the vehicle group and the test article treated animals at 2.5%, 5%, 10%, 25% and 50% (v/v) were normal in size and appearance. Exposure to LLNA-384 2.5%, 5%, 10%, 25% or 50% (v/v) resulted in stimulation indices of 1.8, 1.8, 1.7, 2.8 and 4.3, respectively (Table 3). In addition, the response with 25% and 50% of the test article were both 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 28.3%.

Applicant's summary and conclusion

Interpretation of results:

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

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 50% of LLNA-384 resulted in a stimulation index of 3.0 or greater and hence is considered to have skin sensitizing activity. The EC3 was calculated to be 28.3%.

Executive summary:

To determine the potential of the test article to induce hypersensitivity response in mice, groups of 5 CBA/J 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 LLNA-384, or with the vehicle (DEP). On Day 6,the mice were injected, i.v., with 20µCi 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 were precipitated with 5% trichloroacetic acid (TCA) and the pellets counted in a β-scintillation counter to determine incorporation of the 3 H -thymidine.

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). Ears that appeared wet were observed in all mice treated with the vehicle and 2.5, 5, 10, 25 and 50% of the test article on Days 2-4. Mice treated with the test article at 25 and 50% also had ears that appeared wet on Days 5 and 6. There were no other findings. The mean body weights and body weight changes of the mice treated with LLNA-384 on Days 1 and 6 showed no statistically significant differences. Therefore, administration of the test article did not appear to exert overt toxicity. At termination, the lymph nodes from the mice in the vehicle group and the test article treated animals at 2.5%, 5%, 10%, 25% and 50% (v/v) were normal in size and appearance. Exposure to LLNA-384 2.5%, 5%, 10%, 25% or 50% (v/v) resulted in stimulation indices of 1.8, 1.8, 1.7, 2.8 and 4.3, respectively. In addition, the response with 25% and 50% of the test article were both statistically significant (p<0.001) when the log DPM was compared to the vehicle group. Since the data indicated a positive response and thence is considered to have skin sensitizing actiivty, the EC3 was calculated to be 28.3%.