L-p-mentha-1(6),8-dien-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-08-06 to 30-08-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study and GLP compliant. No justification of the choice of vehicle was given.

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

L-p-mentha-1(6),8-dien-2-one

EC Number:

229-352-5

EC Name:

L-p-mentha-1(6),8-dien-2-one

Cas Number:

6485-40-1

Molecular formula:

C10H14O

IUPAC Name:

(5R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): I-carvone
- Physical state: Pale yellow liquid
- Analytical purity: 99.7%
- Lot/batch No.: 1493525
- Expiration date of the lot/batch: January 2007
- Storage condition of test material: Ambient temperature (under inert gas)

- Specific activity (if radiolabelling): 2.0Ci/mmol
- Locations of the label (if radiolabelling): 3H-methyl thymidine

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca/Ola/Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Oxon, UK.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-17.4g
- Housing: Maximum of 4 per cage, in cages suitable for animals of this strain and weight range,
- Diet (e.g. ad libitum): RM1 diet (Special Diet Services Ltd., Essex, UK) ad libitum.
- Water (e.g. ad libitum): Mains water ad libitum.
- Acclimation period: 5 days

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

Study design: in vivo (LLNA)

Vehicle:

other: 1:3 ethanol/diethylphthlate

Concentration:

Test substance: 2.5, 5, 10, 25 and 50% w/v in 1:3 ethanol/diethylphthlate

No. of animals per dose:

4 animals per dose

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay

- Criteria used to consider a positive response: The lab tests the reliability of the test system every 6 months. Postive control study used for comparison: 19-25th July 2006 (Study No.: GM8020). Hexyl cinnamic aldehyde (Sigma Aldrich) was used as the positive control. The vehicle for the positive control was acetone in olive oil (4:1) The criterion for a positive response is that one of more concentrations of the test substance should elicit a 3-fold or greater increase isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: Test substance (2.5, 5, 10, 25 or 50% w/v) in 25μL vehicle/vehicle alone applied to dorsum of each ear. The procedure was repeated for 3 consecutive days. Three days after the third application, 250μL of phosphate-buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine (2.0Ci/mmol specific activity) was injected into all test and control mice via the tail vein. Five hours later the animals were killed via halothane and the draining auricular lymph node of each ear was excised into PBS.

A single-cell suspension was prepared by gentle mechanical disaggregation through 200 micron-mesh stainless steel gauze.
The cell suspensions were then washed three times by centrifugation with approx. 10mL PBS. and the DNA
precipitated with 3mL 5% trichloroacetic acid (TCA) at 4C overnight. Pellets were re-suspended in 1 mL TCA.

The lymph node suspensions were transferred to scintaillaiton vials and 10 ML scintillant was added prior to β-scintillation using a Apckard Tri-Carb 3100TR Liquid Scintillation Counter.

Statistics:

The EC3 value was calculated by interpolating between two points on the SI axis, one immediately above, and the other immediately below, the SI value of three. The vehicle-treated control value (SI=1) cannot be used for the latter. Where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a,b) and (c,d) respectively, then the EC3 value may be calculated using the following equation :
EC3 = c + [(3-d)/(b-d)] x (a-c).

The quantity applied per squared centimetre was derived from this value, assuming that the area of the mouse ear is 1 cm2 and that 1μL is equiavalent to 1mg.

Results and discussion

Positive control results:

There was no positive control study conducted concurrently. However, the reliability of the test system was confirmed by the most recent positive control assay (Hexyl cinnamic aldehyde; 19-25th July 2006 (Study No.: GM8020; Table 2).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0

Test group / Remarks:

Contorol

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

2.5%

Results 3

Parameter:

SI

Value:

1.5

Test group / Remarks:

5%

Results 4

Parameter:

SI

Value:

2.9

Test group / Remarks:

10%

Results 5

Parameter:

SI

Value:

5

Test group / Remarks:

25%

Results 6

Parameter:

SI

Value:

5.7

Test group / Remarks:

50%

Results 7

Parameter:

EC3

Value:

10.7

Test group / Remarks:

EC3 value 10.7% (2675 μg/cm2)

Any other information on results incl. tables

Table 2:Skin sensitisation potenital of the positive control substance (Hexyl cinnamic aldehyde)

[study run within a six month interval (19-25th July 2006; Study No.: GM8020)]

Concentration of hexylcinnamaldehyde (% w/v)	Number or lymph nodes assayed	Disintegrations per minute (dpm)	dpm per lymph node	Test: control ration (SI)
0 (vehicle only)	8	5939	742	N/A
5	8	10111	1264	1.7
10	8	13747	1718	2.3
25	8	38015	4752	6.4

Vehicle: Acetone in olive oil (4:1)

Applicant's summary and conclusion

Interpretation of results:

sensitising

Conclusions:

The test substance, I-carvone in 1:3 ethanol/diethylphthlate is a skin sensitiser under the conditions of the test with an EC3 value of 10.7% (2675 μg/cm2).

Executive summary:

In a dermal sensitization study (GM8030-REG) with L-carvone (in 1:3 ethanol/diethylphthlate), young adult CBA/Ca/Ola/Hsd mice (4 females) were tested in the Local Lymph Node Assay. The reliability of the test system was confirmed by the most recent positive control assay (Hexyl cinnamic aldehyde; July 2006; Study number: GM8020).

Treament with L-carvone at 2.5, 5, 10, 25 and 50% w/v in 1:3 ethanol/diethylphthlate resulted in resulted in stimulation indices of 1, 1.5, 2.9, 5, and 5.7 respectively. The EC3 value giving rise to a 3 fold increase in lymphocyte proliferation was calculated to be 10.7% (2675 μg/cm2).

In this study, L-carvone is a dermal sensitizer.