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Administrative data

Description of key information

Based on an OECD 429 study (GLP, K1) Clary sage oil is a skin sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-08 October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD guideline 429 with minor deviations: temperature and relative humidity were sometimes outside of the target ranges
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
24th April 2002
Deviations:
yes
Remarks:
temperature and relative humidity were sometimes outside of the target ranges
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 247710
- Date of receipt: 27 September 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature and protected from light
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: 21.2 ± 1.2 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
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
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 = 18.88) were noted. The study was therefore considered valid.
Key result
Parameter:
EC3
Value:
ca. 14.34
Remarks on result:
other: considered as a moderate skin sensitizer
Key result
Parameter:
SI
Value:
1.74
Test group / Remarks:
at the concentration 5%
Key result
Parameter:
SI
Value:
1.45
Test group / Remarks:
at the concentration 10%
Key result
Parameter:
SI
Value:
5.52
Test group / Remarks:
at the concentration 25%
Key result
Parameter:
SI
Value:
8.85
Test group / Remarks:
at the concentration 50%
Key result
Parameter:
SI
Value:
10.73
Test group / Remarks:
at the concentration 100%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
In the proliferation assay, the observed SI values were 1.74, 1.45, 5.52, 8.85 and 10.73 at 5, 10, 25, 50 and 100%, respectively. Refer Table 7.4.1/1 for details.

CELLULAR PROLIFERATION DATA
A dose-related increase in the stimulation index was noted and the threshold positive value of 3 for the stimulation index was exceeded at the concentrations ≥ 25%, leading to a maximum SI value of 10.73 at the concentration of 100%. In the absence of local irritation, the positive lymphoproliferative responses observed at the concentrations ≥25% were attributed to delayed contact hypersensitivity.

EC3 CALCULATION
The EC3 value for the test item was equal to 14.34%.

CLINICAL OBSERVATIONS:
No clinical signs and no mortality were observed during the study.

BODY WEIGHTS
The body weight gain of the treated animals was similar to that of controls.

LOCAL IRRITATION
No cutaneous reactions and no noteworthy increase in ear thickness were observed in the treated groups.

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)

621.65

1081.68

900.49

3430.56

5499.31

6671.63

11736.56

Disintegrations per minute per node (dpm)

77.71

135.21

112.56

428.82

687.41

833.95

1467.07

Stimulation Index (SI)

-

1.74

1.45

5.52

8.85

10.73

18.88

Increase in ear thickness (% between Day 1 and Day 6)

5.52

2.02

4.95

0.00

5.26

6.19

-

EC3 value

-

14.34%

-

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

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

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. No cutaneous reactions and no noteworthy increase in ear thickness were observed in the treated groups. In the proliferation assay, the observed SI values were 1.74, 1.45, 5.52, 8.85 and 10.73 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 14.34%.

The SI of the positive control was 18.88; 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

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. No cutaneous reactions and no noteworthy increase in ear thickness were observed in the treated groups. In the proliferation assay, the observed SI values were 1.74, 1.45, 5.52, 8.85 and 10.73 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 14.34%.

The SI of the positive control was 18.88; therefore this experiment was considered valid.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information the substance is classified as:

- Skin Sens. 1, H317 (May cause an allergic skin reaction) according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

No information was available regarding respiratory sensitisation.