[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-11-2004 to 09-11-2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study - GLP. No information about purity of the test substance and no justification of the choice of vehicle was provided.

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2005

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:

[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one

EC Number:

251-020-3

EC Name:

[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one

Cas Number:

32388-55-9

Molecular formula:

C17H26O

IUPAC Name:

1-((3R,3aR,7R,8aS)-3,6,8,8-tetramethyl-2,3,4,7,8,8a-hexahydro-1H-3a,7-methanoazulen-5-yl)ethanone

Test material form:

other: clear yellow liquid

Details on test material:

- Name of test material (as cited in study report): Acetyl cedrene
- Physical state: clear yellow liquid
- Lot/batch No.: 2004274-0030

- Specific activity (if radiolabelling): 5.0Ci/mmol
- Locations of the label (if radiolabelling): 3H-thymidine


- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbour, ME, USA.
- Age at study initiation: 9-11 weeks at start of dosing
- Weight at study initiation: 19-24 g on Day 1
- Housing: Animals were group housed (5 per cage) upon receipt then 4 per cage when assigned to groups in compliance with National Research Council 'Guide for the Care and Use of Laboratory Animals'.
- Diet (e.g. ad libitum): Certified Rodent Chow 7012C ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2-26.7℃
- Humidity (%): 22-44%
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Study design: in vivo (LLNA)

Vehicle:

other: Diethyl phthalate/ethanol in a 3:1 ratio. No justification provided.

Concentration:

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

No. of animals per dose:

4 animals per dose for test substance

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 reliability of this test has been assessed within a six month interval of the study reported here. Positive control study used for comparison: October 2004 (Study number: 0787MR25.001; Table 5). Hexyl cinnamic aldehyde was used as the positive control. 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: Mice were treated on the dorsal surface of each ear, once per day on Days 1, 2 and 3. On day 6, 250μL of phosphate-buffered saline (PBS) containing 20 μCi of 3H-thymidine (5.0Ci/mmol specific activity) was injected into all test and control mice. Five hours later the animals were killed via CO2 asphyxiation and the lymph nodes were removed and pooled to prepare a single cell suspension. The cell suspensions were then washed twice with PBS and precipitated with 5% trichloroacetic acid (TCA) at 2-8C overnight. Pellets were recovered by centrifugation and re-suspended in 1 mL TCA.

Statistics:

Body weight: ANOVA F-test, Dunnett's test.

EC3 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:

Positive control study used for comparison: There was no positive control study conducted concurrently. However, the reliability of the test system wasconfirmed by the most recent positive control assay (Hexyl cinnamic aldehyde; October 2004 (Study number: 0787MR25.001; Table 5).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.67

Test group / Remarks:

2.5%

Results 2

Parameter:

SI

Value:

1.69

Test group / Remarks:

5%

Results 3

Parameter:

SI

Value:

2.39

Test group / Remarks:

10%

Results 4

Parameter:

SI

Value:

4.72

Test group / Remarks:

25%

Results 5

Parameter:

SI

Value:

25.09

Test group / Remarks:

50%

Results 6

Parameter:

EC3

Value:

13.93

Any other information on results incl. tables

Table 2: Dermal Irritation scores

Mouse No.	Group	Day1		Day 2		Day 3		Day 4		Day 5		Day 6
1	1	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema
2	1	0	0	0	0	0	0	0	0	0	0	0	0
3	1	0	0	0	0	0	0	0	0	0	0	0	0
4	1	0	0	0	0	0	0	0	0	0	0	0	0
5	2	0	0	0	0	0	0	0	0	0	0	0	0
6	2	0	0	0	0	0	0	0	0	0	0	0	0
7	2	0	0	0	0	0	0	0	0	0	0	0	0
8	2	0	0	0	0	0	0	0	0	0	0	0	0
9	3	0	0	0	0	0	0	0	0	0	0	0	0
10	3	0	0	0	0	0	0	0	0	0	0	0	0
11	3	0	0	0	0	0	0	0	0	0	0	0	0
12	3	0	0	0	0	0	0	0	0	0	0	0	0
13	4	0	0	0	0	0	0	0	0	0	0	0	0
14	4	0	0	0	0	0	0	0	0	0	0	0	0
15	4	0	0	0	0	0	0	0	0	0	0	0	0
16	4	0	0	0	0	0	0	0	0	0	0	0	0
17	5	0	0	0	0	0	0	0	0	0	0	0	0
18	5	0	0	0	0	0	0	0	0	0	0	0	0
19	5	0	0	0	0	0	0	0	0	0	0	0	0
20	5	0	0	0	0	0	0	0	0	0	0	0	0
21	6	0	0	0	0	1	0	1	0	1	0	0	0
22	6	0	0	0	0	1	0	1	0	1	0	0	0
23	6	0	0	0	0	1	0	1	0	1	0	0	0
24	6	0	0	0	0	1	0	1	0	1	0	0	0

Table 3: Body weights

Group	Dose	Body weights (g) (mean±sem)		Change in body weight (g) (mean ± sem)
		Day 1	Day 6
1	- (1)	20.5 ± 0.6	20.8 ± 0.5	0.3 ± 0.3
2	3%	21.5 ± 0.6	21.5 ± 1.0	0.0 ± 0.4
3	5%	21.0 ± 0.7	21.5 ± 0.9	0.5 ± 0.3
4	10%	21.0 ± 1.1	22.5 ± 1.0	1.5 ± 0.5
5	25%	20.8 ± 1.0	22.0 ± 0.9	1.3 ± 0.3
6	50%	20.0 ± 0.4	20.5 ± 0.3	0.5 ± 0.3

(1) Diethyl phthalate/ethanol in a ratio of 3:1

Table 4: Local lymph node assay (test substance)

Group	Dose	dpm	SI (Test/control ratio)	Results (2)
1	- (1)	147	-	-
2	2.5%	245	1.67	-
3	5%	248	1.69	-
4	10%	351	2.39	-
5	25%	695	4.72	+
6	50%	3688	25.09	+

(1) Diethyl phthalate/ethanol in a ratio of 3:1

(2) Test/control ration of 3.0 or greater represents a positive result

EC3 = 13.93%

Table 5: Local lymph node assay (positive control)

[study run within a six month interval (October 2004 (Study number: 0787MR25.001)]

Treatment	Dose	dpm (mean±sem)	SI (Test/control ratio)	Results (2)
Vehicle (1)	-	152 ± 11	-	-
HCA	5%	227 ± 45	1.49	-
HCA	15%	428 ± 166*	2.82	-
HCA	35%	524 ± 59**	3.45	+

(1) Diethyl phthalate/ethanol in a ratio of 3:1

(2) Test/control ration of 3.0 or greater represents a positive result

*Statistically sig. difference when the log DPM data of test treated group was compared to vehicle control (p<0.05)

**Statistically sig. difference when the log DPM data of test treated group was compared to vehicle control (p<0.001)

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Based on the criteria of this study, treatment with Acetyl cedrene at 25% and 50% (w/v) resulted in a stimulation index of 3 or greater and hence is consdiered to be a potential skin sensitiser with an EC3 of 13.93.

Executive summary:

In a dermal sensitization study (0787MR25.002) with Methyl Cedryl Ketone (2.5, 5, 10, 25 and 50% w/v in 3:1 Diethyl phthalate/ethanol), young adult female CBA/J mice (4/group) were tested using the local lymph node assay. The reliability of the test system was confirmed by the most recent positive control assay (Hexyl cinnamic aldehyde; October 2004 (Study number: 0787MR25.001)

There was no mortality and all animals appeared normal throughout the study. There were no statistically significant differences observed between any treatment groups with respect to body weight. Very slight erythema was noted in all animals treated with acetyl cedrene (50% w/v) on Days 3 -5. Treatment with Methyl Cedryl Ketone at 2.5%, 5%, 10%, 25% or 50% (w/v) resulted in stimulation indices (SI) of 1.67, 1.69, 2.39, 4.72 and 25.09 respectively. The EC3 was calculated to be 13.93%. In this study, Methyl Cedryl Ketone is a potential skin sensitizer.