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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-05-21 to 2010-07-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the OECD guideline 429 and in compliance with GLP without any deviations.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material (as cited in study report): Beta pinene, Bicyclo[3.1.1]heptane, 6,6-dimethyl-2-methylene / Pin-2(10)-ene
NOTE: Due to an industry habit inherited from the past, the test material was referenced as beta pinene whereas it was actually (-)-beta pinene.
Physical state: colorless to slightly amber coloured liquid
Lot/batch No.: EG12R
Expiration date of the lot/batch: 2011-04-21
Stability under test conditions: to limit the degradation of the products by the oxygen of air, 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.
Storage condition of test material: at 4°C, protected from light and humidity and under nitrogen gas.
Analytical purity: 98.8% (certificate available)
Impurities (identity and concentrations): Beta Phellandrene (0.2%); Myrcene (0.2%); Alpha Pinene (0.1%); Dipentene (0.1%); Camphene (0.1%); Delta 3 carene (0.1%)
Purity test date: 2010-04-22

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: 20.5 g ± 1.3 g
- Housing: in individual crystal polystyrene cages
- 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 (%): 50 ± 20
- 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 (non-LLNA)

Inductionopen allclose all
Route:
other: Not applicable
Vehicle:
other: Not applicable
Concentration / amount:
Not applicable
Challengeopen allclose all
Route:
other: Not applicable
Vehicle:
other: Not applicable
Concentration / amount:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Challenge controls:
Not applicable
Positive control substance(s):
not specified

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
Acetone, Batch No. K38945413830 (Merck, Chelles, France); Olive oil, Batch No.1223873 (Sigma , 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, 5, 10, 25, 50 and 100% 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 (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. No cutaneous reaction was observed at any concentration up to 50%. At the highest test concentration (100%) only, dryness of the skin was observed in one animal. The highest increase in ear thickness (15.38%), observed in one animal treated at the concentration of 100%, was attributed to a slight irritation. Therefore, the test item was not excessively irritant in the preliminary test, whatever the concentration. The highest concentration retained for the main test was therefore the maximal practicable concentration (100%), according to the criteria specified in the International Guidelines.
- 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:
In the positive control group given HCA at the concentration of 25%, 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)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
A dryness of the skin was noted in all animal treated at the concentration of 100% (group 6), associated with an erythema in one of them. The increase in ear thickness measured in group 6 was 21.36%, corresponding to a slightly irritant potential of the test item when used in its original form. A significant lymphoproliferation was noted at tested concentrations of 50% and 100%: the stimulation index was higher than 3 at these two concentrations. In the absence of excessive local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value for the test item Beta pinene is equal to 29% (see table 7.4.1/3).
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.

Table 3: Study results

Groups

Treatment and concentrations

Cell count

Viability (%)

Amount of cells (x106cells)

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

88

16

84.62

8.80

-

8

1286.00

160.75

-

-3.88

-

-

2

Test item 5%

190

18

91.35

19.00

2.16

8

2947.00

368.38

2.29

-1.01

I

29%

3

Test item 10%

48

39

55.17

4.80

0.55

8

1487.00

185.88

1.16

-0.98

I

4

Test item 25%

143

23

86.14

14.30

1.63

8

2869.00

358.63

2.23

-2.02

I

5

Test item 50%

270

40

87.10

27.00

3.07

8

9218.00

1152.25

7.17

-2.83

I

6

Test item 100%

219

41

84.23

21.90

2.49

8

8318.00

1039.75

6.47

21.36

II

7

HCA 25%

622

44

93.39

62.20

7.07

8

18223.00

2277.88

14.17

-

-

-

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:Beta pinene

dpm: disintegration per minute

HCA:α-hexylcinnamaldehyde

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

II: slightly irritant (increase in ear thickness = 10 to 30%)

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

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions of this study, the test item induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Therefore, (-)-beta-pinene is classified as skin sensitizer category 1B according to CLP regulation (EC) No 1272/2008 and as skin sensitizer "Xi, R43: May cause sensitisation by skin contact" according to Directive 67/548/EEC.
Executive summary:

In a dermal sensitization study performed according to OECD guideline 429 and in compliance with the GLP, (-)-beta-pinene in Acetone/Olive oil (4/1, v/v) was administered to CBA/J mice. In the preliminary assay, at the highest tested concentration (100%) only, dryness of the skin was observed in one animal. The highest increase in ear thickness (15.38%), observed in one animal treated at the concentration of 100%, was attributed to a slight irritation. Since the test item was not excessively irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (100%).

Five treated groups of four animals receiving applications of 0.25 µL of the test item to the external surface of botch ears at the concentration of 5, 10, 25, 50 or 100% in the vehicle. The Lymph node proliferative responses were measured as described by Kimber and Dearman (1991).

The positive control (α-hexylcinnamaldehyde) gave acceptable positive results (stimulation index of 14.17).

No clinical signs and no mortality were observed during the study. A dryness of the skin was noted in all animal treated at the concentration of 100%, associated with an erythema in one of them. The increase in ear thickness measured in the 100% group was 21.36%, corresponding to a slightly irritant potential of the test item when used in its original form. A significant lymphoproliferation was noted at tested concentrations of 50% and 100%: the stimulation index was higher than 3 at these two concentrations. In the absence of excessive local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value was 29%.

Under the experimental conditions of this study, (-)-beta-pinene induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Therefore, (-)-beta-pinene is classified as skin sensitizer category 1B according to CLP regulation (EC) No 1272/2008 and as skin sensitizer "Xi, R43: May cause sensitisation by skin contact" according to Directive 67/548/EEC.