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Description of key information

Positive results for skin sensitisation were obtained in a reliable in vivo study with laboratory animals for turpentine (Magnusson, 1969, guinea pig maximisation test). In addition, turpentine was reported to be an extreme sensitiser when tested in humans (Kligman, 1966, Section 7.10.4). Based on the available evidence, it can be concluded that TOPP is a skin sensitiser (Category 1B) according to Regulation (EC) No 1272/2008.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-05-21 to 2010-07-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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)
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 (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°C (+/- 2°C)
- Humidity (%): 50% (+/- 20%)
- Air changes (per hr): approximately 12 cycles per hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: To: no data
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 7.4.1/1
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
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 7.4.1/3). The study was therefore considered valid.
Key result
Parameter:
SI
Value:
2.29
Variability:
91.35
Test group / Remarks:
animals treated with 5 %
Remarks on result:
other: see Remarks
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).
Key result
Parameter:
SI
Value:
1.16
Variability:
55.17 %
Test group / Remarks:
animals treated with 10 %
Remarks on result:
other:
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:
SI
Value:
2.23
Variability:
86.14 %
Test group / Remarks:
animals treated with 25 %
Remarks on result:
other: see Remarks
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:
SI
Value:
7.17
Variability:
87.10 %
Test group / Remarks:
animals treated with 50 %
Remarks on result:
other: see Remarks
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:
SI
Value:
6.47
Variability:
84.23
Test group / Remarks:
animals treated with 100 %
Remarks on result:
other: see Remarks
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 Remarks
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).

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 7.4.1/3: 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

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, the test item Beta pinene induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. Therefore, beta pinene is classified as skin sensitizer in category 1 (H317: May cause an allergic skin reaction) according to the CLP regulation No. 1272/2008 and as skin sensitizer (Xi, R43: May cause sensitisation by skin contact) according to the Directive 67/548/EEC.
Executive summary:

In a dermal sensitization study (No. 36798 TSS) performed according to the OECD guideline No.429 and in compliance with the GLP,pin-2(10)-ene (Beta pinene) (Purity of 98.8%) in Acetone/Olive oil (4/1, v/v) was administered to CBA/J 9 weeks old mice (Janvier). 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 Beta pinene to the external surface of both 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 used was HCA (α-hexylcinnamaldehyde) which presented a Stimulation index of 14.17. 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. 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%.

Under the experimental conditions of this study, the test item Beta pinene induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. Therefore, Beta pinene is classified as skin sensitizer in category 1 (H317: May cause an allergic skin reaction) according to the CLP regulation No. 1272/2008 and as skin sensitizer (Xi, R43: May cause sensitisation by skin contact) according to the Directive 67/548/EC.

This study is considered as acceptable as it satisfied the main criteria of the OECD guideline No. 429.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
no information on age and housing conditions of animals; details on patch material, nature and degree of toxicity, grading system not mentioned
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
The cumulative contact enhancement test (Tsuchiya et al. 1985)
GLP compliance:
no
Type of study:
patch test
Justification for non-LLNA method:
An LLNA study was not performed because there is an existing reliable study for skin sensitisation using the patch test method.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 250-300 g
Route:
epicutaneous, occlusive
Vehicle:
other: olive oil / arachis oil
Concentration / amount:
50%
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: olive oil / arachis oil
Concentration / amount:
Vehicle, 1, 3 and 10%
No. of animals per dose:
12 or 10
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Four
- Exposure period: 24 hours
- Control group: Vehicle only, olive oil
- Site: Scapular region
- Duration: 10 days with an intradermal injection of Freunds complete adjuvant at the third event followed by an open skin test on day 21.
- Concentrations: 50%

B. CHALLENGE EXPOSURE
- Control group: Vehicle only, arachis oil
- Site: Shaven Flanks
- Concentrations: 10, 3 and 1%


Challenge controls:
10 challenge controls
Key result
Reading:
1st reading
Group:
test group
Dose level:
10, 3 and 1%
No. with + reactions:
8
Total no. in group:
12
Clinical observations:
Minimum criterion for positive reaction: erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Group:
test group
Dose level:
10, 3 and 1%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
Minimum criterion for positive reaction: erythema
Remarks on result:
positive indication of skin sensitisation
Group:
positive control
Remarks on result:
other: positive control data not reported
Group:
negative control
Remarks on result:
other: negative control data not reported

None

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, 3-carene was found to be potent skin-sensitiser.
Executive summary:

3-carene was tested for skin sensitisation in female Dunkin-Hartley guinea pigs. Groups of guinea pigs (10 or 12/group) received four occluded dermal exposure for 24 hours in scapular region of 50% carene followed by challenge dose on the shaven flanks with 10, 3, 1% and vehicle.

 

In the first set of experiments 8 out of 12 and in second, 7 out of 10 animals showed presence of erythema which was considered as positive reaction.

Under the test conditions, 3-carene was found to be potent skin-sensitiser.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1969
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no data on housing conditions and individual weight of animals; no data on nature and degree of effects observed
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
no data on housing conditions and individual weight of animals; no data on nature and degree of effects observed
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An LLNA study was not performed because there is an existing reliable study for skin sensitisation using the Guinea Pig Maximisation test method.
Species:
guinea pig
Strain:
other: albino
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 300-500 g
Route:
intradermal and epicutaneous
Vehicle:
petrolatum
Concentration / amount:
- Induction: 5% (Intradermal) and 25% by weight (topical)
- Challenge: 20% by weight
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
- Induction: 5% (Intradermal) and 25% by weight (topical)
- Challenge: 20% by weight
No. of animals per dose:
10 female
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- Exposure period: One week (first stage); 48 hours (second stage)
- Site: Shoulder
- Concentrations: 5% and 25% by weight (intradermal and topical, respectively)

B. CHALLENGE EXPOSURE
- Site: Flank
- Concentrations: 20% by weight
- Evaluation (hr after challenge): 24

C. Maximization grading:
Sensitization rate: 0-8% - grade I- Weak; 9-28% - grade II- Mild; 29-64% - grade III- moderate; 65-80% - grade IV- strong; 81-100%- grade V- extreme
Challenge controls:
The animals were challenged two weeks after the topical induction by applying 20% by weight of turpentine oil in petrolatum to the flank for 24 hours under a 4 cm strip of Blenderm.
Positive control substance(s):
not specified
Positive control results:
No data
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
20% by weight
No. with + reactions:
16
Total no. in group:
25
Remarks on result:
positive indication of skin sensitisation
Group:
positive control
Remarks on result:
other: positive control data not reported
Group:
negative control
Remarks on result:
other: negative control data not reported

None

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, turpentine oil is considered to be as skin sensitizer.
Executive summary:

In a skin sensitization study performed following the method similar to OECD guideline 406, sensitization potential of turpentine oil was assessed by guinea pig maximization test. Group of female albino guinea pigs were induced with 5% (intradermal injection) and 25% (topical) by weight of turpentine oil, followed by a challenge dose of 20% by weight of turpentine oil in petrolatum to the flank on clipped skin using an occlusive patch held in place for 24 hours. The challenge site was evaluated 24 hours after removal of the patch.

16 out of 25 animals tested showed the signs of sensitization during the study period which corresponds to 64% of sensitization rate.

Under the test conditions, turpentine oil is considered as a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In vitro and in vivo skin sensitisation data are available for Turpentine Oil itself and for a number of its constituents. These are summarised as follows:

A guinea pig maximisation test (Magnusson, 1969) is available for turpentine of unknown composition (identified as CAS 8006-64-2) which gave a clear positive result for skin sensitisation. In addition, a human patch test (Kligman, 1966) showed that turpentine (unspecified composition) is an extreme skin sensitiser in humans.

Additional data for constituents have also been identified as follows:

β-Pinene (CAS 127-91-3):

An LLNA assay is available for β-pinene (OECD 429, GLP), in which significant lymphoproliferation was noted at tested concentrations of 50% and 100% (Pelcot, C 2010). The stimulation index was higher than 3 at these two concentrations, therefore classification as skin sensitizer in category 1 B is appropriate. 

δ-3-Carene (CAS 13466-78-9):

An in vivo sensitisation assay is available for δ-3-carene (similar to OECD 406)(Lastbom, L, 2003). After four topical inductions over a period of ten days using 50% test substance in olive oil, with an intradermal injection of Freund’s complete adjuvant at the third event, Guinea pigs were challenged with 1, 3 and 5% δ-3-carene. A positive reaction was observed in approximately 70% of animals, so it is concluded that classification as skin sensitizer in Category 1 B is appropriate.

 

Alpha-Terpineol (CAS 98-55-5): Not sensitising (non-guideline study, similar to EU Method B.42)

Dimethyl disulfide (CAS 624-92-0): Sensitising Category 1B (OECD 429)

References:

Pelcot, C 2010: Evaluation of skin sensitization potential in mice using the local lymph node assay (LLNA) (study report), Testing laboratory: CIT, Evreux, France, Report no: 36798 TSS. Owner company; Les Dérivés Résiniques et Terpéniques (DRT), 30 rue Gambetta, BP 206, 40105 DAX Cedex, France, Report date: Aug 19, 2010

Lastbom L, Boman A, Johnsson S, Camner P and Ryrfeldt A 2003: Increased airway responsiveness of a common fragrance component, 3-carene, after skin sensitisation-a study in isolated guinea pig lungs (publication), Toxicol. Lett. 145: 189-196. Testing laboratory: Division of Inhalation Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Turpentine (CAS 8006-64-2) is formally classified in Annex VI of EC Regulation 1272/2008/EC as follows:

According to the criteria of EU Directive 67/548/EEC: Xi; R43 "May cause sensitization by skin contact".

According to the criteria of 1272/2008/EC: Skin Sensitiser Category 1B.

The available in vivo test data for turpentine and its constituents β-pinene and δ-3 -carene, as well as evidence from a study in humans, indicate that the substance is an extreme sensitiser, supporting the official classification.

No data are available regarding respiratory sensitisation.