(-)-pin-2(10)-ene

Skin irritation / corrosion

Administrative data

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.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

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. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(-)-pin-2(10)-ene

EC Number:

242-060-2

EC Name:

(-)-pin-2(10)-ene

Cas Number:

18172-67-3

Molecular formula:

C10H16

IUPAC Name:

(1S,5S)-6,6-dimethyl-2-methylidenebicyclo[3.1.1]heptane

Details on test material:

Name of test material (as cited in study report): beta-pinene
NOTE: Due to an industry habit inherited from the past, the test material was referenced as beta-pinene whereas it was actually (-)-beta-pinene.
Physical state: colourless translucent liquid
Analytical purity: 98.8 %
Impurities (identity and concentrations): beta phellandrene 0.2%, myrcene 0.2%, alpha-pinene 0.1%, dipentene 0.1%, camphene 0.1%, delta-3-carene 0.1%
Purity test date: 22 April 2010
Lot/batch No.: EG12R
Expiration date of the lot/batch: 21 April 2011
Storage condition of test material: kept in refrigerator under nitrogen flow and protected from light and from humidity

Test animals

Species:

human

Details on test animals or test system 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

Test system

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

Results and discussion

In vitro

Results

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)

In vivo

Irritant / corrosive response data:

No data

Other effects:

No data

Any other information on results incl. tables

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

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, (-)-beta-pinene is classified as "R38, irritating to skin" according to Directive 67/548/EEC and as skin irritant category 2 in CLP Regulation (EC) No 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 "R38 irritating to skin" according to Directive 67/548/EEC and as skin irritant category 2 according to CLP Regulation (EC) No 1272/2008.