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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:
22-30 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline 439 without any deviation
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane
EC Number:
938-945-4
Molecular formula:
Not applicable
IUPAC Name:
Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Terpinolene multiconstituent
- Physical state: Yellow liquid
- Analytical purity: 66.9 %
- Composition of test material (%): Terpineols (4.4 %), alpha pinene (1.1 %), alpha fenchene (0.2 %), camphene (1.0 %), alpha phellandrene (0.5 %), alpha terpinene (2.7 %), cineol 1.4 (20.5 %), d-limonene (9.1 %), l-limonene (9.1 %), beta phellandrene (0.2 %), paracymene (0.7 %), cineol 1.8 (14.6 %), gamma terpinene (3.6%), terpinolene (31.8 %) and others (0.5 %)
- Lot/batch No.: 123238
- Purity test date: 17 October 2011
- Date of receipt: 16 April 2012
- Expiration date of the lot/batch: 29 September 2012
- Storage condition of test material: Stored at 6 ± 3 °C in darkness

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:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: negative control: PBS+; positive control: 5 % (w/v) SDS solution
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration: Test material: undiluted; negative control: PBS+; positive control: 5 % (w/v) SDS solution
Duration of treatment / exposure:
15 ± 0.5 min
Observation period:
Post-treatment incubation period: 42 ± 1 h
Number of animals:
Three epidermis units were used per test material, positive and negative controls
Details on study design:
TEST SYSTEM:
- Cell system used: EPISKIN® kit: three-dimensional human epidermis model
- Source: SKINETHIC Laboratories (Lyon, France)
- All biological components of the epidermis and the kit culture medium have been tested for the absence of viruses, bacteria and mycoplasma. The quality was assessed by an MTT cytotoxicity test and by histological examination (by SKINETHIC).

METHODOLOGY:
- Non-specific MTT reduction: 10 µL of the test item was added to each well of a 12-wells plate containing 2 mL of MTT solution (0.3 mg/mL) and colour of the solution was checked after incubation for 3 h ± 5 min at 37 ± 1 °C, 5 ± 1 % CO2, 95 ± 5% humidity (CO2 incubator).
- Staining power: 10 µL of the test item was agitated with 90 µL of mineral oil for 15 ± 0.5 min at room temperature and colour of the solution was checked.
- MTT conversion assay: The epidermis units were transferred from the kit into maintenance medium filled wells and pre-incubated for 24 ± 3 h at 37 ± 2 °C. After pre-incubation, the test materials (10 µL) were applied topically to the epidermal model (three epidermis units per test material, positive and negative controls) for 15 ± 0.5 min at room temperature. After rinsing with phosphate buffered saline, the epidermises were then incubated at 37 ± 2 °C for 42 ± 1 h (CO2 incubator). Aliquots of culture media were kept frozen (-20°C) for cytokine (IL-1α) further measurements. The viability was assessed by incubating the tissues for 3 h ± 0.5 min with MTT solution in a 12 well plate (0.3 mg/mL; 2 mL per well). The formazan precipitated was then extracted using acidified isopropanol (0.5 mL) and quantified spectrophotometrically at 570 nm using 96 well plates (200 μL/well). For each treated tissue the viability was expressed as a % relative to negative control tissues (mean).
- IL-1α assay: Standard or samples (200 µL) were added to each well containing 50 µL of diluent and incubated for 2 h ± 15 min at room temperature. After washing with buffer, 200 µL of conjugate IL-1α was added to each well and incubated for 1 h ± 10 min at room temperature. The wells were washed again and added with 200 µL of substrate solution. After incubation for 20 ± 5 min at room temperature, optical density was recorded at 450 nm on a microplate reader. Protein levels of IL-1α in the supernatant were expressed as pg/mL.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: relative viability %
Value:
63.5
Remarks on result:
other:
Remarks:
Basis: mean. Time point: exposure: 15 min. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:
Negative control:
- Mean optical density: 0.855
Positive control:
- Viability %: 25.1 ± 3.9
- IL-1 α mean concentration: 250.1 pg/mL
Test material:
- Viability %: 63.5 ± 5.4
- IL-1 α mean concentration: 184.9 pg/mL

- See tables 7.3.1/1 and 7.3.1/2 for data
Other effects:
- Non-specific MTT reduction: TERPINOLENE MULTICONSTITUENT did not interact with MTT.
- Staining power: TERPINOLENE MULTICONSTITUENT had no staining power on living epidermis.

Any other information on results incl. tables

Table 7.3.1/1: MTT conversion assay

OD1

OD2

Mean

Standard Deviation

Viability %

Mean Viability %

Standard deviation

Negative control

Epidermis 1

0.827

0.878

0.853

0.036

99.7

100

3.7

Epidermis 2

0.891

0.885

0.888

0.004

103.8

Epidermis 3

0.828

0.822

0.825

0.004

96.5

Mean

/

/

0.855

/

/

Positive control

Epidermis 1

0.242

0.254

0.248

0.008

29

25.1

3.9

Epidermis 2

0.233

0.195

0.214

0.027

25

Epidermis 3

0.206

0.158

0.182

0.034

21.3

P1

Epidermis 1

0.506

0.551

0.529

0.032

61.8

63.5

5.4

Epidermis 2

0.603

0.586

0.595

0.012

69.5

Epidermis 3

0.511

0.501

0.506

0.007

59.2

O.D. Optical Density

Table 7.3.1/2: Interleukine 1 alpha (IL-1α) assay

OD

Concentration IL-1α (pg/mL)

Mean concentration IL-1α (pg/mL)

Standard deviation

CV

IL-1α final concentration (pg/mL) (treated/untreated)

Epidermis 1

Epidermis 2

Epidermis 3

Epidermis 1

Epidermis 2

Epidermis 3

Negative control

0.022

0.031

0.023

-7.2

-6.0

-7.1

-6.8

0.69

-10.3%

/

Positive control

1.803

1.799

1.801

243.6

243.1

243.3

243.3

0.28

0.10%

250.1

P1

1.353

1.35

1.311

180.2

179.8

174.3

178.1

3.3

1.90%

184.9

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, cell viability was > 50 % on reconstructed human epidermis model therefore Terpinolene multiconstituent was considered as non-irritant according to the criteria of Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).
Executive summary:

In an in vitro skin irritation study performed according to the OECD Guideline 439 and in compliance with GLP, 10 µL of the test item, TERPINOLENE MULTICONSTITUENT was applied topically to reconstructed human epidermis model (3 epidermis units/dose) for 15 ± 0.5 min at room temperature. After rinsing with phosphate buffered saline (PBS), epidermises were incubated at 37 ± 2 °C for 42 ± 1 h (CO2 incubator). Aliquots of culture media were kept frozen (-20°C) for cytokine (IL-1α) further measurements. The viability was assessed by incubating the tissues for 3 h ± 0.5 min with MTT solution in a 12 well plate (0.3 mg/mL; 2 mL per well). The formazan precipitated was then extracted using acidified isopropanol (0.5 mL) and quantified spectrophotometrically at 570 nm using 96 well plates (200 μL/well). SDS 5% (w/v) and PBS-treated epidermis were used as positive and negative controls, respectively.

Mean relative cell viability for the test item was 63.5 ± 5.4 %. Mean cell viability for the positive control was 25.1 ± 3.9 % and optical density value for the negative control epidermis was 0.855 and thus confirmed the validity of the test. Mean IL-1 α concentrations of test item and positive controls were recorded to be 184.9 and 250.1 pg/mL, respectively.

Under the test conditions, cell viability was > 50 % on reconstructed human epidermis model therefore Terpinolene multiconstituent was considered as non-irritant according to the criteria of Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

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