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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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): alpha-pinene
- Physical state: colourless translucent liquid
- Analytical purity: 96.6 %
- Impurities (identity and concentrations): camphene 1.7%, beta-pinene 1.1%, alpha fenchene 0.1%
- Purity test date: 22 April 2010
- Lot/batch No.: 108348
- Expiration date of the lot/batch: 19 April 2011
- Storage condition of test material: 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 vivo

Results
Irritation parameter:
other: viability % (MTT assay)
Basis:
mean
Time point:
other: 15 min
Score:
39.6
Reversibility:
no data
Remarks on result:
other: ± 5.6
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) = 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

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, alpha pinene is classified as irritating to skin, R38, according to the criteria of Annex VI to the Directive 67/548/EEC and category 2, H319 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 skin, R38, according to the criteria of Annex VI to the Directive 67/548/EEC and category 2, H319 in CLP Regulation (EC) N° 1272 /2008.