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Toxicological information

Skin irritation / corrosion

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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-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
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:
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 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:
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.