Registration Dossier

Administrative data

Description of key information

Three major constituents of TOPP met the EU criteria for classification as skin irritants based on percentage cellular viability <50% in Episkinin vitrostudies (Maillet, 2010a, b and c).

Two major constituents of TOPP did not meet EU criteria for classification as eye irritants in GLP studies conducted in accordance with OECD Test Guideline 405 (Colas, 2010a and b). Turpentine (unspecified composition) was reported to cause adverse ocular effects following sub-conjunctival injection in a peer-reviewed publication.

TOPP also contains low concentrations of the sulfur-containing substances methyl mercaptan (typically 0.06%), dimethyl sulfide (typically 1.2%) and dimethyl disulfide (0.32%). The maximum total combined concentration of these constituents reported in commercial samples is 6% by weight expressed as sulfur. None of these is classified as irritant or corrosive in Annex VI of EC Regulation 1272/2008/EC. Data reported in IUCLID 2000 indicate that in OECD guideline studies dimethyl disulfide (CAS 624-92-0) did not meet the criteria for classification as skin or eye irritant. Slight effects are reported in non-guideline studies for dimethyl sulfide, but these are unlikely to meet the criteria for classification. Methyl mercaptan (CAS 74-93-1) is reported to be corrosive in IUCLID 2000, based on a test with sodium methyl mercaptide (pH 9.78). However, the validity of this read-across is questionable since the observed corrosive effects are most likely due to the high pH, which would not be relevant for neutral methyl mercaptan present in TOPP.

Overall, the sulfur-containing species present in TOPP would not contribute to a more severe classification for skin and eye irritation than the existing one.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from June 22 to July 27, 2010
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:
equivalent or similar to
Guideline:
other: ECVAM protocol version 1.8 of February 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals and environmental conditions:
Three-dimensional human epidermis model, supplied by SkinEthic Laboratories, Nice, France constituted by:
- a collagen type I matrix, coated with type IV collagen
- a differentiated and stratified epidermis model from human keratinocytes, obtained after 13-day culture period.
All biological components of the epidermis and the kit culture medium have been tested for the absence of viruses, bacteria and mycoplasma
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 µL
Duration of treatment / exposure:
15 ± 0.5 min
Number of animals:
3 epidermis/product
Details on study design:
Before the beginning of the study, the non-specific MTT reduction by the test item was checked by incubating MTT solution and 10 µL test item for 3 hours ± 30 minutes and checking the colour of the solution.

Application of the test item and control:
- negative control: 10 µL of PBS+ to each epidermis surface
- positive control: 10 µL of SDS solution at 5% (w/v) in distilled water; after 7 minutes contact time, an intermediate re-spreading is done
- test substance: 10 µL of the test item, as supplied
- contact timepoint for all products: 15 ± 0.5 minutes at room temperature

Rinsing:
- epidermis rinsed thoroughly with 25 mL of PBS+
- remaining product removed with a cotton-bud

Incubation: plates incubated during 42 ± 1 hours in CO2 incubator with maintenance medium

MTT assay:
- epidermis incubated for 3 hours ± 5 minutes in MTT solution in the CO2 incubator
- formazan extraction in 500 µL of acidic isopropanol stored at 4 hours ± 30 minutes at room temperature, protected from light or during 70 ± 5 hours at 4 °C
- measurement of OD at 570 nm
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.

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:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the test conditions, beta-pinene is classified as irritating to the skin, R38, according to the criteria of Annex VI to the Directive 67/548/EEC and category 2 in CLP Regulation (EC) N° (1272-2008).
Executive summary:

A GLP study conducted in vitro 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 test item was applied directly on epidermis for 15 min on 3 epidermis. 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 38.5 ± 3.5. As the percentage of viability is ≤ 50 %, the test item is considered to be irritating for skin. Under the test conditions, beta-pinene is classified as irritating to the skin, R38, according to the criteria of Annex VI to the Directive 67/548/EEC and category 2 in CLP Regulation (EC) N° (1272-2008).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from June 22 to July 27, 2010
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:
equivalent or similar to
Guideline:
other: ECVAM protocol version 1.8 of February 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals and environmental conditions:
Three-dimensional human epidermis model, supplied by SkinEthic Laboratories, Nice, France constituted by:
- a collagen type I matrix, coated with type IV collagen
- a differentiated and stratified epidermis model from human keratinocytes, obtained after 13-day culture period.
All biological components of the epidermis and the kit culture medium have been tested for the absence of viruses, bacteria and mycoplasma
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 µL
Duration of treatment / exposure:
15 ± 0.5 min
Number of animals:
3 epidermis/product
Details on study design:
Before the beginning of the study, the non-specific MTT reduction by the test item was checked by incubating MTT solution and 10 µL test item for 3 hours ± 30 minutes and checking the colour of the solution.

Application of the test item and control:
- negative control: 10 µL of PBS+ to each epidermis surface
- positive control: 10 µL of SDS solution at 5% (w/v) in distilled water; after 7 minutes contact time, an intermediate re-spreading is done
- test substance: 10 µL of the test item, as supplied
- contact timepoint for all products: 15 ± 0.5 minutes at room temperature

Rinsing:
- epidermis rinsed thoroughly with 25 mL of PBS+
- remaining product removed with a cotton-bud

Incubation: plates incubated during 42 ± 1 hours in CO2 incubator with maintenance medium

MTT assay:
- epidermis incubated for 3 hours ± 5 minutes in MTT solution in the CO2 incubator
- formazan extraction in 500 µL of acidic isopropanol stored at 4 hours ± 30 minutes at room temperature, protected from light or during 70 ± 5 hours at 4 °C
- measurement of OD at 570 nm
Irritation / corrosion parameter:
other: other: viability % (MTT assay)
Value:
29.8
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Reversibility: no data. Remarks: ± 1.3.

Negative control (PBS+): mean OD = 0.821

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

Test item: % viability (MTT assay) = 29.8 ± 1.3

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

0.249

0.265

0.257

0.011

31.3

29.8

0.013

Epidermis 2

0.236

0.236

0.236

0.000

28.8

Epidermis 3

0.230

0.254

0.242

0.017

29.5

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the test conditions, delta-3-carene is classified as irritating to the skin, R38, according to the criteria of Annex VI to the Directive 67/548/EEC and category 2 in CLP Regulation (EC) N° (1272-2008).
Executive summary:

A GLP study conducted in vitro with human epidermis model EPISKIN was performed to assess the irritancy potential of delta-3-carene similarly to ECVAM protocol version 1.8 of February 2009. 10 µL of test item was applied directly on epidermis for 15 min on 3 epidermis. 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 29.8 ± 1.3. As the percentage of cell viability is ≤ 50 %, the test item is considered to be irritating for skin. Under the test conditions, delta-3-carene is classified as irritating to the skin, R38, according to the criteria of Annex VI to the Directive 67/548/EEC and category 2 in CLP Regulation (EC) N° (1272-2008).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from June 22 to July 27, 2010
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:
equivalent or similar to
Guideline:
other: ECVAM protocol version 1.8 of February 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals and environmental conditions:
Three-dimensional human epidermis model, supplied by SkinEthic Laboratories, Nice, France constituted by:
- a collagen type I matrix, coated with type IV collagen
- a differentiated and stratified epidermis model from human keratinocytes, obtained after 13-day culture period.
All biological components of the epidermis and the kit culture medium have been tested for the absence of viruses, bacteria and mycoplasma
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 µL
Duration of treatment / exposure:
15 ± 0.5 min
Number of animals:
3 epidermis/product
Details on study design:
Before the beginning of the study, the non-specific MTT reduction by the test item was checked by incubating MTT solution and 10 µL test item for 3 hours ± 30 minutes and checking the colour of the solution.

Application of the test item and control:
- negative control: 10 µL of PBS+ to each epidermis surface
- positive control: 10 µL of SDS solution at 5% (w/v) in distilled water; after 7 minutes contact time, an intermediate re-spreading is done
- test substance: 10 µL of the test item, as supplied
- contact timepoint for all products: 15 ± 0.5 minutes at room temperature

Rinsing:
- epidermis rinsed thoroughly with 25 mL of PBS+
- remaining product removed with a cotton-bud

Incubation: plates incubated during 42 ± 1 hours in CO2 incubator with maintenance medium

MTT assay:
- epidermis incubated for 3 hours ± 5 minutes in MTT solution in the CO2 incubator
- formazan extraction in 500 µL of acidic isopropanol stored at 4 hours ± 30 minutes at room temperature, protected from light or during 70 ± 5 hours at 4 °C
- measurement of OD at 570 nm
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.

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:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the test conditions, alpha-pinene is classified as irritating to the skin, R38, according to the criteria of Annex VI to the Directive 67/548/EEC and category 2 in CLP Regulation (EC) N° (1272-2008).
Executive summary:

A GLP study conducted in vitro 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 test item was applied directly on epidermis for 15 min on 3 epidermis. 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 39.6 ± 5.6. As the percentage of viability is ≤ 50 %, the test item is considered to be irritating for skin. Under the test conditions, alpha-pinene is classified as irritating to the skin, R38, according to the criteria of Annex VI to the Directive 67/548/EEC and category 2 in CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: The information is taken from a peer reviewed publication. No study details are available therefore reliability cannot be assigned.
Qualifier:
no guideline followed
Principles of method if other than guideline:
No information available on method.
GLP compliance:
not specified
Species:
other: no data
Strain:
not specified
Details on test animals or tissues and environmental conditions:
No information available.
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
No information.
Duration of treatment / exposure:
No information.
Observation period (in vivo):
No information.
Number of animals or in vitro replicates:
No information.
Details on study design:
No information.
Remarks on result:
other: No irritation scores reported.
Irritant / corrosive response data:
No irritation scores reported.
Other effects:
Subconjunctival injection of turpentine in one case caused Phthisis bulbi. Injection into anterior chamber of animals causes fibrinopurulent inflammation with corneal opacification from endothelial injury and infiltration of leukocytes.
Interpretation of results:
irritating
Conclusions:
Adverse ocular effects (no scores provided) are reported in a peer reviewed publication. Turpentine is classified as an eye irritant in Annex VI of Regulation 1272/2008/EC.
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From May 10 to 24, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no data on tool used to asses score
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
no data on tool used to asses score
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
no data on tool used to asses score
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Elevage de Gerome, Quartier Labaste, France
- Age at study initiation: 11 or 13 weeks
- Weight at study initiation: 2.08-3.07 kg
- Housing: Each animal housed in individual box
- Diet: Foodstuff (SDS - C15); ad libitum
- Water: Tap water (public distribution system); ad libitum
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70%
- Air changes (per hour): 15/hour
- Photoperiod: 12 hours dark / 12 hours light
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume with unit): 0.1 mL
Duration of treatment / exposure:
No washing was done
Observation period (in vivo):
Eight days
Number of animals or in vitro replicates:
Three males
Details on study design:
SCORING SYSTEM:

Chemosis
No swelling …0
Slight swelling, including the nictitating membrane…1
Swelling with eversion of the eyelid...2
Swelling with eyelid half-closed…3
Swelling with eyelid more than half-closed…4

Discharge
No discharge…0
Slight discharge (normal slight secretion in the inner corner not to be taken into account)…1
Discharge with moistening of the eyelid and neighboring hairs…2
Discharge with moistening of the eyelids and large areas around the eye…3

Redness
Blood vessel normal…0
Vessels significantly more prominent than normal…1
Vessels individually distinguishably with difficulty
Generally red coloration….2
Generally deep red coloration….3

Iris
Normal…0
Iris significantly more wrinkled than normal, congestion, swelling of the iris which continues to react to light, even slowly…1
No reaction to light, haemorrhage, significant damage (any or all of these characteristics)…2

Cornea: degree of opacity
No modification visible either directly or after instillation of fluorescein (no loss of glint or polish)…0
Translucent areas (diffuse or disseminated), iris details clearly visible…1
Early identifiable translucent area, iris details slightly obscured…2
Opalescent area, no iris details visible, pupil outline scarcely distinguishable…3
Total corneal opacity, completely obscuring the iris and pupil…4

Cornea: extent of opacity
Opaque area present but covering one quarter or less…1
Between one quarter and half…2
Between half and three quarters…3
Between three quarters and the entire surface…4
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 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:
24/48/72 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:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 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:
24/48/72 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:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 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:
24/48/72 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:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 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:
GHS criteria not met
Conclusions:
Under the test conditions, BETA-PINENE is not classified as irritating to the eye according to the criteria of Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).
Executive summary:

In an eye irritation study conducted according to the 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.

 

Under the test conditions, BETA-PINENE is not classified as irritating to the eye according to the criteria of Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From May 10 to 25, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no data on tool used to asses score
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
no data on tool used to asses score
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
no data on tool used to asses score
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Elevage de Gerome, Quartier Labaste, France
- Age at study initiation: 11 or 13 weeks
- Weight at study initiation: 2.32-3.18 kg
- Housing: Each animal housed in individual box
- Diet: Foodstuff (SDS - C15); ad libitum
- Water: Tap water (public distribution system); ad libitum
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70%
- Air changes (per hour): 15/hour
- Photoperiod: 12 hours dark / 12 hours light
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume with unit): 0.1 mL
Duration of treatment / exposure:
No washing was done
Observation period (in vivo):
Eight days
Number of animals or in vitro replicates:
Three females
Details on study design:
SCORING SYSTEM:

Chemosis
No swelling …0
Slight swelling, including the nictitating membrane…1
Swelling with eversion of the eyelid...2
Swelling with eyelid half-closed…3
Swelling with eyelid more than half-closed…4

Discharge
No discharge…0
Slight discharge (normal slight secretion in the inner corner not to be taken into account)…1
Discharge with moistening of the eyelid and neighboring hairs…2
Discharge with moistening of the eyelids and large areas around the eye…3

Redness
Blood vessel normal…0
Vessels significantly more prominent than normal…1
Vessels individually distinguishably with difficulty
Generally red coloration….2
Generally deep red coloration….3

Iris
Normal…0
Iris significantly more wrinkled than normal, congestion, swelling of the iris which continues to react to light, even slowly…1
No reaction to light, haemorrhage, significant damage (any or all of these characteristics)…2

Cornea: degree of opacity
No modification visible either directly or after instillation of fluorescein (no loss of glint or polish)…0
Translucent areas (diffuse or disseminated), iris details clearly visible…1
Early identifiable translucent area, iris details slightly obscured…2
Opalescent area, no iris details visible, pupil outline scarcely distinguishable…3
Total corneal opacity, completely obscuring the iris and pupil…4

Cornea: extent of opacity
Opaque area present but covering one quarter or less…1
Between one quarter and half…2
Between half and three quarters…3
Between three quarters and the entire surface…4
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 8 days
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 8 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 8 days
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 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:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
- Moderate redness of the conjunctivae associated with 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

3

2

0

0

24 hours

2

2

0

0

48 hours

2

2

0

0

72 hours

2

2

0

0

Day 4

2

2

0

0

Day 7

0

1

0

0

Day 8

0

0

0

0

Mean (24, 48 and 72 hours)

2

2

0

0

2

1 hour

3

24 hours

2

2

0

0

48 hours

1

1

0

0

72 hours

1

1

0

0

Day 4

1

1

0

0

Day 7

1

1

0

0

Day 8

0

0

0

0

Mean (24, 48 and 72 hours)

1.33

1.33

0

0

3

1 hour

3

2

0

0

24 hours

2

2

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

1.33

0

0

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, DELTA 3 CARENE is not classified as irritating to the eye according to the criteria of Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).
Executive summary:

In an eye irritation study conducted according to the OECD Guideline 405 and in compliance with GLP, three female rabbits of the New Zealand White strain were exposed to 0.1 mL of undiluted DELTA 3 CARENE 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 DELTA 3 CARENE resulted in moderate redness of the conjunctivae associated with 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; 2, 1.33, 1.33 for conjunctivae score and 2, 1.33, 1 for chemosis score.

 

Under the test conditions, DELTA 3 CARENE is not classified as irritating to the eye according to the criteria of Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The registered substance, TOPP, includes Crude Sulfate Turpentine (CST) and Wood Turpentine (WT). A detailed description of the manufacturing processes and composition of TOPP is given in Section 1.2.1 and Section 1.2.2 respectively. The principal constituents of TOPP are α-pinene (10 - 85%), β-pinene (0 - 40%), δ-3-carene (0 - 40%) and dipentene (0 - 20%). Lower concentrations (up to around 5% each) of other terpenes are present. These terpene constituents are also present in varying proportions in other forms of turpentine, all of which are captured in the general description of CAS Number 8006-64-2).

Skin irritation

No in vitro or in vivo skin irritation data are available for TOPP itself. However, data are available for a number of its constituents. These are summarised as follows:

α-Pinene (CAS 80-56-8), β-pinene (CAS 127-91-3) and δ-3-carene (CAS 13466-78-9)

Recent, reliable in vitro skin irritation studies are available for three of the major constituents of TOPP, α-pinene (CAS 80-56-8), β-pinene (CAS 127-91-3) and δ-3-carene (CAS 13466-78-9) (Maillet, 2010a, b and c). Together these constituents typically comprise ca. 78% by weight of the registration substance. The studies were conducted according to an appropriate test protocol (ECVAM) and in compliance with GLP, and were therefore assigned Klimisch score 1. In all three cases, the study results indicated that the relevant test substance met the criteria for classification as a skin irritant, based on percentage cellular viability <50%. Based on the available weight of evidence for these main constituents, TOPP is therefore expected to be a skin irritant.

Information on skin irritation has also been identified for other constituents and is summarised below, although this does not impact the conclusion for classification.

Terpinolene/Isoterpinolene (CAS 586-62-9): Not irritant (OECD 439)

1,8-Cineole (CAS 470-82-6): Not irritant (OECD 439)

Alpha-Terpineol (CAS 98-55-5): Irritant (OECD 404)

Dimethyl disulfide (CAS 624-92-0): Irritant (OECD 404)

Eye irritation

No in vitro or in vivo eye irritation data are available for TOPP itself. However, data are available for a number of its constituents. These are summarised as follows:

β-Pinene (CAS 127-91-3) and δ-3-carene (CAS 13466-78-9)

Recent, reliable in vivo eye irritation studies are available for two of the major constituents of TOPP, β-pinene (CAS 127-91-3) and δ-3-carene (CAS 13466-78-9) (Colas, 2010a and b). Together, these constituents typically comprise 21% by weight of the registration substance. The studies were conducted according to OECD Test Guideline 405 and in compliance with GLP, and were therefore assigned Klimisch score 1. In both cases, although adverse effects were observed (chemosis and conjunctival redness), these were not sufficient to trigger classification for eye irritation according to EU critera. The REACH dissemination dossier for α-pinene[1] uses read-across from the result with β-pinene as key data for the eye irritation endpoint.

Published eye irritation data for turpentine (unspecified composition, CAS 8006-64-2) are cited in a publication for which reliability could not be assigned (Grant, 1974, cited in HSDB). Subconjunctival injection of turpentine in one case caused Phthisis bulbi (shrinkage and atrophy of the eyeball). Injection into anterior chamber of animals causes fibrinopurulent inflammation with corneal opacification from endothelial injury and infiltration of leukocytes. The method used is not relevant for classification for eye irritation according to Regulation (EC) No 1272/2008.

Based on the available weight of evidence for the main constituents, TOPP is therefore not expected to be an eye irritant.

Information on skin irritation has also been identified for other constituents and is summarised below, although this does not impact the conclusion for classification.

Terpinolene/Iso terpinolene (CAS 586-62-9): Not irritant (OECD 405)

1,8-Cineole (CAS 470-82-6): Not irritant (OECD 437)

Dimethyl disulfide (CAS 624-92-0): Irritant (OECD 405)


[1] Accessed 7th November 2016

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; R36/38 "Irritating to eyes and skin".

According to the criteria of 1272/2008/EC: Skin Irritant Category 2 and Eye Irritant Category 2.

The available in vitro skin irritation studies for the constituents α-pinene, β-pinene and δ-3-carene support the classification for skin irritation.

The available in vivo eye irritation studies for the constituents β-pinene and δ-3-carene suggest that while irritant responses were observed in test animals, these were not sufficient to trigger classification for eye irritation according to EU criteria (Colas, 2010). Adverse ocular effects following exposure to turpentine are reported in a secondary citation of a peer reviewed publication (Grant, 1974). Insufficient information is available to determine scores relative to EU criteria. However, the available evidence is not sufficient to refute the official classification; therefore, TOPP is also classified as an eye irritant.