7-methyl-3-methyleneocta-1,6-diene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-07 to 2010-06-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to the OECD guideline 429 and in compliance with GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

On day 1, the body weight of two animals was lower than 18 g (17.3 g). This minor deviation was not considered to have compromised the validity or integrity of the study.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

7-methyl-3-methyleneocta-1,6-diene

EC Number:

204-622-5

EC Name:

7-methyl-3-methyleneocta-1,6-diene

Cas Number:

123-35-3

Molecular formula:

C10H16

IUPAC Name:

7-methyl-3-methylideneocta-1,6-diene

impurity 1

Reference substance name:

1-methylene-4-(1-methylvinyl)cyclohexane

EC Number:

207-895-9

EC Name:

1-methylene-4-(1-methylvinyl)cyclohexane

Cas Number:

499-97-8

Molecular formula:

C10H16

IUPAC Name:

1-isopropenyl-4-methylenecyclohexane

impurity 2

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-isopropenyl-1-methylcyclohexene

impurity 3

Reference substance name:

(S)-p-mentha-1,8-diene

EC Number:

227-815-6

EC Name:

(S)-p-mentha-1,8-diene

Cas Number:

5989-54-8

Molecular formula:

C10H16

IUPAC Name:

(4S)-isopropenyl-1-methylcyclohexene

impurity 4

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No. 104775
Purity: 93%
Name of test material (as cited in study report): MYRCENE (CAS No. 123-35-3)
Physical state: colourless - slightly amber liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source : Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 9 weeks old
- Weight at study initiation: 19.4 ± 0.8 g
- Housing: in individual crystal polystyrene cages (22 cm x 8.5 cm x 8 cm)
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30-70
- Air changes (per hr): approximately 12 cycles per hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Remarks:

Acetone, Batch No. K38945413830 (Merck, Chelles, France); Olive oil, Batch Nos. 058K0684 (Sigma , Saint-Quentin-Fallavier, France) and 1223873 (Fluka , Saint-Quentin-Fallavier, France).

Concentration:

For the preliminary test the concentrations were 10, 25, 50 and 100% of the test item.
For the main test the concentrations were 0, 2.5, 5, 10, 25 and 50% of the test item.

No. of animals per dose:

For the preliminary test: 2 females/dose (no controls): Right and left ear were treated with different concentrations.
For the main test: 4 females/dose, 4 females for the negative control and 4 females for the positive control
See details on table 1

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: the test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at all tested dilutions (2.5-50%).
- Irritation: for 3 consecutive days, the animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear). Measurement of the ear thickness (using a micrometer) was performed each day before treatment and 72 hours after the last application. An increase in ear thickness was recorded at the concentration of 100%, showing the irritant potential of the test item at this concentration. The highest concentration retained for the main test was therefore 50%.
- Lymph node proliferation response: not applicable.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Lymph node cell proliferative responses were measured as described by Kimber and Dearman (1991).
- Criteria used to consider a positive response: The results were expressed as disintegration per minute (dpm) per group. Stimulation indices (SI) were calculated according to the following formula: SI = dpm of treated group / dpm of control group. The test item was considered as a skin sensitizer when the SI for a dose group is higher than or equal to 3. Other relevant criteria such as cellularity (amount of cells in treated group compared to the amount in control vehicle group), radioactivity levels and ear thickness were also taken into account for the interpretation of results.


TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared in the vehicle at the chosen concentrations. The test item dosage forms were prepared within the 3 hours before the end of use, and kept at room temperature and protected from light until use. The test item was kept in a hermetically closed flask between each opening. Openings were limited as much as possible, but were necessary during the preparation and the animal procedures. On days 1, 2 and 3, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

Positive control substance(s):

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

Statistics:

no data

Results and discussion

Positive control results:

The mean cell viability in the vehicle group was higher than 70% and the threshold positive value of 3 for the SI was reached in the positive control group (see table 3). The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 0.66

Test group / Remarks:

concentration 2.5%

Remarks on result:

other: see details in table 3

Results 2

Key result

Parameter:

SI

Value:

ca. 2.59

Test group / Remarks:

concentration 25%

Remarks on result:

other: see details in table 3

Results 3

Key result

Parameter:

SI

Value:

ca. 2.79

Test group / Remarks:

concentration: 10%

Remarks on result:

other: see details in table 3

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See details in Table 3.

Any other information on results incl. tables

Neither mortality nor clinical signs were observed during the study. The body weight gain of the treated animals was similar to that of the control animals. No cutaneaous reactions and no notable increase in ear thickness were observed at any of the tested concentrations.

Table 3: Study results

Groups	Treatment and concentrations	Cell count		Viability (%)	Amount of cells (x10^6cells)	Cellularity index	Number of nodes per group	dpm per group	dpm per node	Stimulation index (SI)	Increase in ear thickness (% between day1 and day 6)	Irritation level	EC3value
		viable	dead
1	Vehicle	86	19	81.90	8.60	-	8	637.51	79.69	-	6.52	-	-
2	Test item 2.5%	76	4	95.00	7.60	0.88	8	423.72	52.97	0.66	3.06	I	NA
3	Test item 5%	138	5	96.50	13.80	1.60	8	611.42	76.43	0.96	4.04	I
4	Test item 10%	81	20	80.20	8.10	0.94	8	500.47	62.56	0.79	7.45	I
5	Test item 25%	234	17	93.23	23.40	2.72	8	1654.19	206.77	2.59	5.26	I
6	Test item 50%	187	26	87.79	18.70	2.17	8	1132.76	141.60	1.78	2.04	I
7	HCA 25%	271	29	90.33	27.10	3.15	8	2653.43	331.68	4.16	-	-	-

Viability= (viable cells/(viable cells+dead cells))*100

Cellularity index=amount of cells (x10⁶cells) in the treated groups/ amount of cells (x10⁶cells) in the vehicle groups

Stimulation index=dpm of treated group/dpm of control group

Vehicle: acetone/olive oil

Test item: Myrcene

NA: Not applicable

dpm: disintegration per minute

HCA:α-hexylcinnamaldehyde

I: non-irritant (increase in ear thickness < 10%)

EC3 value: Theoretical concentration resulting in a SI value of 3

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Test item myrcene did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay. Therefore, myrcene is not classified as skin sensitiser.

Executive summary:

In a dermal sensitization study performed according to Guideline OECD 429 and in compliance with GLP, myrcene in Acetone/Olive oil (4/1, v/v) was administered to CBA/J 9 weeks old mice. Since the test item was irritant in the preliminary test at the concentration of 100%, the highest tested concentration retained for the main test was 50%.

Five treated groups of four animals received applications of 25 μL of test item myrcene to the external surface of both ears at the concentration of 2.5, 5, 10, 25, or 50 % in the vehicle. The lymph node proliferative responses were measured as described by Kimber and Dearman (1991).

The positive control used was HCA (α-hexylcinnamaldehyde) which presented a stimulation index of 4.16. Therefore, the positive control gave acceptable positive results and the study can be considered valid.

No clinical signs and no mortality were observed during the study. Furthermore, no skin irritation was noted following the application of the test item in the main test. The SI was lower than 3 in all groups treated whatever the concentration of the test item.

Therefore, test item myrcene did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay and is not classified as skin sensitiser.