Lavender, Lavandula hybrida, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD guideline 429 without any deviation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

November 2000,

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Linalool

EC Number:

201-134-4

EC Name:

Linalool

Cas Number:

78-70-6

Molecular formula:

C10H18O

IUPAC Name:

3,7-dimethylocta-1,6-dien-3-ol

Constituent 2

Reference substance name:

Linalyl acetate

EC Number:

204-116-4

EC Name:

Linalyl acetate

Cas Number:

115-95-7

Molecular formula:

C12H20O2

IUPAC Name:

1,5-dimethyl-1-vinylhex-4-en-1-yl acetate

Constituent 3

Reference substance name:

Bornan-2-one

EC Number:

200-945-0

EC Name:

Bornan-2-one

Cas Number:

76-22-2

Molecular formula:

C10H16O

IUPAC Name:

1,7,7-trimethylbicyclo[2.2.1]heptan-2-one

Constituent 4

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Constituent 5

Reference substance name:

4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene

Cas Number:

118-65-0

Molecular formula:

C15H24

IUPAC Name:

4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene

Constituent 6

Reference substance name:

(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

EC Number:

207-353-1

EC Name:

(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Cas Number:

464-45-9

Molecular formula:

C10H18O

IUPAC Name:

endo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Constituent 7

Reference substance name:

p-menth-1-en-4-ol

EC Number:

209-235-5

EC Name:

p-menth-1-en-4-ol

Cas Number:

562-74-3

Molecular formula:

C10H18O

IUPAC Name:

1-isopropyl-4-methylcyclohex-3-en-1-ol

Constituent 8

Reference substance name:

2-isopropenyl-5-methylhex-4-enyl acetate

EC Number:

247-327-7

EC Name:

2-isopropenyl-5-methylhex-4-enyl acetate

Cas Number:

25905-14-0

Molecular formula:

C12H20O2

IUPAC Name:

5-methyl-2-(prop-1-en-2-yl)hex-4-en-1-yl acetate

Constituent 9

Reference substance name:

7,11-dimethyl-3-methylenedodeca-1,6,10-triene

EC Number:

278-628-1

EC Name:

7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Cas Number:

77129-48-7

Molecular formula:

C15H24

IUPAC Name:

7,11-dimethyl-3-methylidenedodeca-1,6,10-triene

Constituent 10

Reference substance name:

p-menth-1-en-8-ol

EC Number:

202-680-6

EC Name:

p-menth-1-en-8-ol

Cas Number:

98-55-5

Molecular formula:

C10H18O

IUPAC Name:

alpha,alpha-4-trimethyl-3-cyclohexene-1-methanol

Constituent 11

Reference substance name:

(Z)-3,7-dimethylocta-1,3,6,-triene

EC Number:

222-081-3

EC Name:

(Z)-3,7-dimethylocta-1,3,6,-triene

Cas Number:

3338-55-4

Molecular formula:

C10H16

IUPAC Name:

(3Z)-3,7-dimethylocta-1,3,6,-triene

Constituent 12

Reference substance name:

2-isopropenyl-5-methylhex-4-en-1-ol

EC Number:

261-264-2

EC Name:

2-isopropenyl-5-methylhex-4-en-1-ol

Cas Number:

58461-27-1

Molecular formula:

C10H18O

IUPAC Name:

5-methyl-2-(prop-1-en-2-yl)hex-4-en-1-ol

Constituent 13

Reference substance name:

Dipentene

EC Number:

205-341-0

EC Name:

Dipentene

Cas Number:

138-86-3

Molecular formula:

C10H16

IUPAC Name:

4-isopropenyl-1-methylcyclohexene

Constituent 14

Reference substance name:

(E)-3,7-dimethylocta-1,3,6-triene

EC Number:

223-241-5

EC Name:

(E)-3,7-dimethylocta-1,3,6-triene

Cas Number:

3779-61-1

Molecular formula:

C10H16

IUPAC Name:

(3E)-3,7-dimethylocta-1,3,6-triene

Constituent 15

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

Constituent 16

Reference substance name:

(1Z,6Z)-1-methyl-5-methylidene-8-propan-2-ylcyclodeca-1,6-diene

Cas Number:

37839-63-7

Molecular formula:

C15H24

IUPAC Name:

(1Z,6Z)-1-methyl-5-methylidene-8-propan-2-ylcyclodeca-1,6-diene

Constituent 17

Reference substance name:

Geraniol

EC Number:

203-377-1

EC Name:

Geraniol

Cas Number:

106-24-1

Molecular formula:

C10H18O

IUPAC Name:

(2E)-3,7-dimethylocta-2,6-dien-1-ol

Constituent 18

Reference substance name:

Hexyl butyrate

EC Number:

220-136-6

EC Name:

Hexyl butyrate

Cas Number:

2639-63-6

Molecular formula:

C10H20O2

IUPAC Name:

hexyl butanoate

Constituent 19

Reference substance name:

Pin-2(3)-ene

EC Number:

201-291-9

EC Name:

Pin-2(3)-ene

Cas Number:

80-56-8

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Constituent 20

Reference substance name:

Pin-2(10)-ene

EC Number:

204-872-5

EC Name:

Pin-2(10)-ene

Cas Number:

127-91-3

Molecular formula:

C10H16

IUPAC Name:

6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Constituent 21

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

Constituent 22

Reference substance name:

Octan-3-one

EC Number:

203-423-0

EC Name:

Octan-3-one

Cas Number:

106-68-3

Molecular formula:

C8H16O

IUPAC Name:

octan-3-one

Constituent 23

Reference substance name:

p-mentha-1(7),2-diene

EC Number:

209-081-9

EC Name:

p-mentha-1(7),2-diene

Cas Number:

555-10-2

Molecular formula:

C10H16

IUPAC Name:

3-methylidene-6-propan-2-ylcyclohexene

Constituent 24

Reference substance name:

3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

EC Number:

236-719-3

EC Name:

3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

Cas Number:

13466-78-9

Molecular formula:

C10H16

IUPAC Name:

3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

Test material form:

liquid

Details on test material:

Name: Lavandin Grosso HE
Appearance: colourless liquid
Batch number: échantillon du 25/03/02
Density: 0.8935 at 20°C
Storage conditions: keep away from light and humidity.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: échantillon du 25/03/02
- Date of receipt: 28 March 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: ca. 9 weeks
- Weight at study initiation: 21.0 ± 1.1 g
- Housing: Animals were housed individually in disposable crystal polystyrene cages.
- Diet: A04 C pelleted diet (UAR, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water: Tap water (filtered using a 0.22 µm filter) contained in bottles, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 30-70%
- Air changes: ca. 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 hours dark / 12 hours light

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 5, 10, 25, 50 and 100% v/v

No. of animals per dose:

4 females

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the SI for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION: 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. On Day 6, all animals of all groups received a single intravenous injection of 250 μL of 0.9% NaCl containing 20 μCi of 3H-TdR (specific activity of 25 Ci/mmol), via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
For each experimental group, a single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 mL of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of Trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three mL of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting. The results were expressed as disintegrations/mn (dpm) per group.

Stimulation Indices (SI) were calculated according to the following formula: SI = dpm of treated group / dpm of control group

Calculation of the EC3 value (theoretical concentration of the test item resulting in a SI value of 3) was performed on the basis of a dose-effect response.

Positive control substance(s):

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

Statistics:

Not applicable

Results and discussion

Positive control results:

In the positive control group given HCA at the concentration of 25%, an increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 10.86) were noted. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

6.74

Remarks on result:

other: at the concentration of 100%

Results 2

Key result

Parameter:

EC3

Value:

31

Results 3

Parameter:

SI

Value:

4.58

Test group / Remarks:

at the concentration of 50%

Results 4

Parameter:

SI

Value:

3.39

Test group / Remarks:

at the concentration of 25%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
In the proliferation assay, the observed SI values were 1.04; 1.36; 3.39; 4.58 and 6.74 at 5, 10, 25, 50 and 100%, respectively. Refer Table 7.4.1/1 for details.

Any other information on results incl. tables

Table 7.4.1/1: Skin sensitization – results

 

Concentrations (%)	Vehicle control (Acetone/Olive oil)	5	10	25	50	100	Positive control (HCA at 25%)
No. of nodes per group	8	8	8	8	8	8	8
Disintegrations per minute per group (dpm)	796.70	828.14	1080.70	2697.52	3648.65	5366.85	7573.66
Disintegrations per minute per node (dpm)	99.59	103.52	135.09	337.19	456.08	670.86	1081.95
Stimulation Index (SI)	-	1.04	1.36	3.39	4.58	6.74	10.86
Increase in ear thickness (% between Day 1 and Day 6)	0.00	0.98	0.00	1.96	-0.99	0.96	-
EC3 value	-	31%					-

 

Stimulation Indices, SI = dpm of treated group / dpm of control group

EC3 value = theoretical concentration resulting in a SI value of 3

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the test conditions, the test item was considered as a moderate skin sensitizer (i.e. EC3 >2%) and need to be classified as "Category 1 and sub-category 1B" according to the Regulation (EC) N° 1272/2008 and according to the Globally Harmonised System of classification and labelling of chemicals (GHS). The hazard statement "H317: may cause an allergic skin reaction" with the symbol “exclamation mark” and signal word "Warning" are required.

Executive summary:

In a Local Lymph Node Assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of test item at concentrations of 5, 10, 25, 50 and 100% v/v to the dorsal surface of both ears for three consecutive days(Days 1, 2 and 3). One negative control group of four animals received the vehicle (mixture acetone/olive oil (4/1)) and one positive control group received the reference item, α-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%. After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (Day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on Days 1, 2, 3 and 6.

No mortality and no clinical signs were observed during the study. Except for dryness of the skin recorded on Day 6 in 2/4 animals given the test item undiluted, no cutaneous reactions and no noteworthy increase in ear thickness were observed during the study. In the proliferation assay, the observed SI values were 1.04; 1.36; 3.39; 4.58 and 6.74  at 5, 10, 25, 50 and 100% respectively. A dose-related increase in the stimulation index was noted and the threshold positive value for the SI was exceeded at the concentrations ≥ 25%. In the absence of local irritation, the positive lymphoproliferative responses observed at the concentrations ≥25% were attributed to delayed contact hypersensitivity. The EC3 value for the test item was equal to 31%.

The SI of the positive control was 10.86; therefore this experiment was considered valid.

Under these test conditions, the test item was considered as a moderate skin sensitizer (i.e. EC3 >2%) and need to be classified as "Category 1 and sub-category 1B" according to the Regulation (EC) N° 1272/2008 and according to the Globally Harmonised System of classification and labelling of chemicals (GHS). The hazard statement "H317: may cause an allergic skin reaction" with the symbol “exclamation mark” and signal word "Warning" are required.