Registration Dossier

Administrative data

Description of key information

(-)-alpha-pinene is classified as irritating to skin, category 2 according to CLP Regulation (EC) n° 1272/2008.

No classification as eye irritant or serious eye damage according to CLP Regulation (EC) n° 1272 /2008 is required for (-)-alpha-pinene.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from June 22 to July 27, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well conducted and in compliance with GLP.
Reason / purpose:
read-across: supporting information
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:
Value:
39.6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Reversibility: no data. Remarks: ± 5.6. (migrated information)
Irritant / corrosive response data:
No data
Other effects:
No data

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

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
October 2015
Species:
human
Strain:
other: reconstructed human Cornea-like Epithelium
Details on test animals or tissues and environmental conditions:
EpiOcular TM tissue Model OCL-212-ver2.0 supplied by MatTek
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test item alpha-pinene multiconstituent was applied as supplied at the dose of 50 µL.
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
A 12-minute post exposure immersion period at room temperature and a 1-hour and 58-minute post exposure incubation at standard culture conditions.
Number of animals or in vitro replicates:
2 replicates
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 multiconstituent 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 multiconstituent 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:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Information on skin irritation:

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. Therefore, (-)-alpha-pinene is classified as irritating to skin, category 2 according to CLP Regulation (EC) n° 1272/2008.

Information on eye irritation:

In a GLP study conducted according to 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 multiconstituent 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.

No classification as eye irritant or serious eye damage according to CLP Regulation (EC) n° 1272 /2008 is required for (-)-alpha-pinene.

Justification for classification or non-classification

According to GLP studies performed on alpha-pinene multiconstituent, (-)-alpha-pinene is classified as irritating to skin category 2 (H315) according to Regulation (EC) n° 1272/2008. The substance is not classified for eye irritation according to Regulation (EC) n° 1272/2008.