Gum turpentine oil, consisting of the volatile fraction resulting from the distillation of oleoresin collected by tapping softwoods from pinacea derivatives (genus: pinus). It consists of terpenes, mainly monoterpenes such as alpha-pinene and beta-pinene.

Endpoint summary

Description of key information

In GLP in vitro studies using Episkin model, alpha pinene and (-)-beta pinene lead to a cell viability of 39.6 ± 5.6, 38.5 ± 3.5 and 29.8 ± 1.3 %, respectively.

In a GLP in vitro study and a in vivo GLP study, alpha-pinene and respectively beta-pinene showed no irritating properties. However, in a GLP study conducted according to OECD Guideline 405, camphene had irritating properties in rabbit eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Beta-Pinene is a main constituent of UVCB substance gum tupentine oil. Therefore, data on beta-pinene can be used for extrapolation to gum turpentine oil. See read-across justification document in section 13.

Reason / purpose for cross-reference:

read-across source

Vehicle:

unchanged (no vehicle)

Irritation / corrosion parameter:

other: other: viability % (MTT assay)

Value:

38.5

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 min. Reversibility: no data. Remarks: ± 3.5. (migrated information)

Negative control (PBS+): mean OD = 0.821

Positive control (5% SDS): % viability (MTT assay) = 18.7 ± 3.0

Test item: % viability (MTT assay) = 38.5 ± 3.5

Table 1: MTT conversion assay in living epidermis

		O.D. 1	O.D. 2	Mean	Standard deviation	Viability %	Mean Viability %	Standard deviation
Negative control	Epidermis 1	0.768	0.808	0.788	0.028	96.0	100	0.044
	Epidermis 2	0.855	0.864	0.860	0.006	104.7
	Epidermis 3	0.819	0.811	0.815	0.006	99.3
Positive control	Epidermis 1	0.124	0.145	0.135	0.015	16.4	18.7	0.030
	Epidermis 2	0.158	0.131	0.145	0.019	17.6
	Epidermis 3	0.191	0.172	0.182	0.013	22.1
Test item	Epidermis 1	0.359	0.293	0.326	0.047	39.7	38.5	0.035
	Epidermis 2	0.301	0.267	0.284	0.024	34.6
	Epidermis 3	0.358	0.318	0.338	0.028	41.2

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The percentage of viability is ≤ 50% and (-)-beta-pinene is classified as irritating to skin, R38, according to the criteria of Directive 67/548/EEC and in category 2 according to CLP Regulation (EC) N° 1272/2008.

Executive summary:

An in vitro GLP study conducted with human epidermis model EPISKIN was performed to assess the irritancy potential of (-)-beta-pinene similarly to ECVAM protocol version 1.8 of February 2009. 10 µL of the test item was applied directly on 3 epidermis for 15 min. Positive control was 10 µL of 5% (w/v) SDS solution and negative control was 10 µL of PBS (each tested on 3 epidermis). MTT conversion assay was peformed to evaluate the percentage of cellular viability of the epidermis. Positive control had a percentage of cell viability of 18.7± 3.0 and test item had a percentage of cell viability of 38.5 ± 3.5. As the percentage of viability is ≤ 50%, the test item is considered to be irritating for skin.

Therefore, (-)-beta-pinene is classified as irritating to skin, R38, according to the criteria of Directive 67/548/EEC and in category 2 according to CLP Regulation (EC) n° 1272/2008.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Alpha-Pinene is a main constituent of UVCB substance gum tupentine oil. Therefore, data on alpha-pinene can be used for extrapolation to gum turpentine oil. See read-across justification document in section 13.

Reason / purpose for cross-reference:

read-across source

Irritation / corrosion parameter:

other: other: viability % (MTT assay)

Value:

39.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 min. Reversibility: no data. Remarks: ± 5.6. (migrated information)

Negative control (PBS+): mean OD = 0.821

Positive control (5% SDS): % viability (MTT assay) = 18.7 ± 3.0

Test item: % viability (MTT assay) = 39.6 ± 5.6

Table 1: MTT conversion assay in living epidermis

		OD 1	OD 2	Mean	Standard deviation	Viability %	Mean Viability %	Standard deviation
Negative control	Epidermis 1	0.768	0.808	0.788	0.028	96.0	100	0.044
	Epidermis 2	0.855	0.864	0.860	0.006	104.7
	Epidermis 3	0.819	0.811	0.815	0.006	99.3
Positive control	Epidermis 1	0.124	0.145	0.135	0.015	16.4	18.7	0.030
	Epidermis 2	0.158	0.131	0.145	0.019	17.6
	Epidermis 3	0.191	0.172	0.182	0.013	22.1
Test item	Epidermis 1	0.327	0.310	0.319	0.012	38.8	39.6	0.056
	Epidermis 2	0.365	0.382	0.374	0.012	45.5
	Epidermis 3	0.273	0.292	0.283	0.013	34.4

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The percentage of viability is ≤ 50 % and alpha-pinene is classified as irritating to skin, R38, according to Directive 67/548/EEC and in category 2 according to CLP Regulation (EC) N° 1272/2008.

Executive summary:

An in vitro GLP study conducted with human epidermis model EPISKIN was performed to assess the irritancy potential of alpha-pinene similarly to ECVAM protocol version 1.8 of February 2009. 10 µL of the test item was applied directly on 3 epidermis for 15 min. Positive control was 10 µL of 5% (w/v) SDS solution and negative control was 10 µL of PBS (each tested on 3 epidermis). MTT conversion assay was peformed to evaluate the percentage of cellular viability of the epidermis. Positive control had a percentage of cell viability of 18.7 ± 3.0 and test item had a percentage of cell viability of 39.6 ± 5.6. As the percentage of viability is ≤ 50 %, the test item is considered to be irritating for skin.

Therefore, alpha-pinene is classified as irritating to skin, R38, according to Directive 67/548/EEC and in category 2 according to CLP Regulation (EC) n° 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Beta-Pinene is a main constituent of UVCB substance gum tupentine oil. Therefore, data on beta-pinene can be used for extrapolation to gum turpentine oil. See read-across justification document in section 13.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 8 days

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 3 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 4 days

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 3 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritant / corrosive response data:

- Moderate redness of the conjunctivae associated with moderate to severe chemosis was noted in all treated animals after 1 hour of instillation; irritation completely resolved within 8 days.
- See table 1 for more details

Other effects:

No data

Table 1: Individual and mean scores of conjunctivae, iris and cornea

 

Animal	Time after treatment	Conjunctivae		Iris	Cornea
		Chemosis	Redness	Lesion	Opacity
1	1 hour	2	2	0	0
	24 hours	1	1	0	0
	48 hours	1	1	0	0
	72 hours	2	1	0	0
	Day 4	2	1	0	0
	Day 7	1	1	0	0
	Day 8	0	0	0	0
	Mean (24, 48 and 72 hours)	1.3	1	0	0
2	1 hour	3	2	0	0
	24 hours	2	1	0	0
	48 hours	1	1	0	0
	72 hours	0	1	0	0
	Day 4	0	0	0	0
	Mean (24, 48 and 72 hours)	1	1	0	0
3	1 hour	3	2	0	0
	24 hours	2	2	0	0
	48 hours	1	2	0	0
	72 hours	0	2	0	0
	Day 4	0	1	0	0
	Day 7	0	0	0	0
	Mean (24, 48 and 72 hours)	1	2	0	0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

beta-Pinene is not classified as irritating to eyes according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Executive summary:

In an eye irritation study conducted according to OECD Guideline 405 and in compliance with GLP, three male rabbits of the New Zealand White strain were exposed to 0.1 mL of undiluted beta-pinene in one of the eyes while the other eye corresponded to the control. The eyes were examined for irritation scores at 1 hour and 1, 2, 3, 4, 7 and 8 days after dosing.

 

Instillation of beta-pinene resulted in moderate redness of the conjunctivae associated with moderate to severe chemosis in all treated animals after 1 hour of instillation. The irritation completely resolved within 8 days. Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 0, 0, 0 for cornea score; 0, 0, 0 for iris score; 1, 1, 2 for conjunctivae score and 1.3, 1, 1 for chemosis score.

 

Therefore, beta-pinene is not classified as irritating to eyes according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Camphene is one of the minor constituents of UVCB substance gum turpentine oil. Therefore, data on camphene can be used for extrapolation to gum turpentine oil. See read-across justification document in section 13.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

0.3

Max. score:

2

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

2.3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

2.3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean individual

Time point:

other: 24, 48 and 72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritant / corrosive response data:

See table 1

Other effects:

See table 1

Table 1: individual scores and observations

Time after application	1 h	24 h	48 h	72 h	7 days
Animal number	1 2 3	1 2 3	1 2 3	1 2 3	1 2 3
Chemosis	3 3 2	3 2 1	2 2 1	1 1 0	0 0 0
Conjunctival redness	3 3 3	3 3 3	2 3 2	2 3 2	0 0 0
Iris	1 1 1	1 1 1	1 1 0	0 0 0	0 0 0
Cornea	1 1 1	2 1 1	1 1 1	1 0 0	0 0 0
Fluorescein test		3 2 1		1 0 0	0 0 0
Ocular discharge: - Clear, colorless -white and slimy   Red iris	X X X     X X X	X X X   X X X	X X

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Camphene is classified as ‘Irritating to eyes (Category 2)’ according to CLP Regulation (EC) n° 1272/2008 but not classified according to the criteria of Directive 67/548/EEC.

Executive summary:

In an eye irritation study conducted according to the OECD Guideline 405 and in compliance with GLP, three rabbits of the New Zealand White strain were exposed to 100 mg of undiluted camphene in one of the eyes while the other eye corresponded to the control. The eyes were examined for irritation scores at 1 hour and 1, 2, 3, and 7 days after application. Examination and scoring after instillation of fluoresceine was assessed at 24 h, 72 h and 7 days after test substance application.

 

Application of camphene resulted in severe redness of the conjunctivae associated with slight to moderate chemosis in all treated animals. The irritation completely resolved within 7 days. Mean individual scores at 24, 48 and 72 hours after exposure for the 3 animals were 1.3, 0.7, 0.7 for cornea score; 0.7, 0.7, 0.3 for iris score; 2.3, 3, 2.3 for conjunctivae score and 2, 1.7, 0.7 for chemosis score.

Therefore, camphene is classified as ‘Irritating to eyes (Category 2)’ according to CLP Regulation (EC) n° 1272/2008 but not classified according to the criteria of Directive 67/548/EEC.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Alpha-Pinene is a main constituent of UVCB substance gum tupentine oil. Therefore, data on alpha-pinene can be used for extrapolation to gum turpentine oil. See read-across justification document in section 13.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

other: percent tissue viability

Value:

80.39

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

percent tissue viability = 31.99%

Table 1:

INDIVIDUAL AND AVERAGE VALUES OF OD AFTER 30 MINUTES EXPOSURE

	Tissue	OD	Mean OD/disc	Mean OD/ product	Viability %	Mean viability %	Difference of viability %
Negative control	1	1.035 1.023 0.961	1.006	1.079	93.28	100.00	13.44
	2	1.158 1.128 1.167	1.151		106.72
Positive control	3	0.367 0.351 0.357	0.358	0.345	31.19	31.99	2.41
	4	0.335 0.333 0.328	0.332		30.78
Test item	7	0.896 0.879 0.862	0.879	0.867	81.50	80.39	2.33
	8	0.873 0.853 0.841	0.855		79.28

OD: optical density

 

Interpretation of results:

GHS criteria not met

Conclusions:

alpha-Pinene does not need classification as eye irritant or serious eye damage according to Regulation (EC) n° 1272/2008 and UN GHS.

Executive summary:

In a GLP study conducted according to the OECD 492 guideline the irritant potential of alpha-pinene multiconstituent was evaluated. The method is based on viability of Reconstructed Human Cornea-like Epithelium (RhCE) when exposed to the test substance. The mean percent tissue viability of the RhCE replicates treated with test item alpha-pinene was 80.39% versus 31.99% in the positive control (Methyl acetate). In conclusion, in accordance with Regulation (EC) n° 1272/2008 and UN GHS, the test item does not require classification as eye irritant or serious eye damage. No hazard statement or signal word is required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

In GLP in vitro studies using Episkin model, exposure of epidermis to alpha pinene and (-)-beta pinene for 15 min lead to a cell viability, measured by MTT conversion, of 39.6 ± 5.6 and 38.5 ± 3.5 respectively.

In a GLP study conducted according to OECD Guideline 492, application of alpha-pinene resulted in mean percent tissue viability of the RhCE replicates of 80.39%. In a GLP study conducted according to OECD Guideline 405 application of beta-pinene resulted in moderate redness of the conjunctivae associated with moderate to severe chemosis in all treated animals after 1 hour of instillation. The irritation completely resolved within 8 days. However, in a GLP study conducted according to OECD Guideline 405, application of camphene resulted in severe redness of the conjunctivae associated with slight to moderate chemosis in all treated animals. The irritation completely resolved within 7 days.

Justification for classification or non-classification

As the percentage of cell viability obtained in Episkin model was ≤ 50 % for alpha pinene and beta pinene, they are considered to be irritating for skin. Based on a read across approach, gum turpentine oil is classified as irritating to the skin, R38, according to the criteria of Directive 67/548/EEC and in category 2 according to CLP Regulation (EC) n° 1272 /2008.

As camphene was found to be eye irritating in an in vivo study, gum turpentine oil is classified as ‘Irritating to eyes (Category 2)’ according to CLP Regulation (EC) n° 1272/2008 but not classified according to the criteria of Directive 67/548/EEC.