Reaction mass of Terpenes and Terpenoids, turpentine-oil, limonene fraction, 1-methyl-4-(1-methylethenyl)cyclohexene and turpentine-oil beta-pinene fraction terpenes, dimers and Terpenes and Terpenoids, turpentine-oil, limonene fraction, 1-methyl-4-(1-methylethenyl)cyclohexene and turpentine-oil beta-pinene fraction terpenes, trimers

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/08/2012 to 27/08/2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers

IUPAC Name:

Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers

Constituent 2

Reference substance name:

Terpenes and terpenoids, turpentine-oil, α-pinene fraction, oligomers

EC Number:

500-245-8

EC Name:

Terpenes and terpenoids, turpentine-oil, α-pinene fraction, oligomers

Cas Number:

70750-57-1

Test material form:

other: extremely viscous pale amber coloured liquid

Details on test material:

- Name of test material (as cited in study report): Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers
- Substance type: UVCB
- Physical state: extremely viscous pale amber coloured liquid
- Analytical purity: 100%
- Lot/batch No.: 0910002624
- Expiration date of the lot/batch: indefinite
- Storage condition of test material:room temperature in the dark

Specific details on test material used for the study:

Test Material: Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers
CAS Number: 70750-57-1
EC Number: 500-245-8
Batch Number: 0910002624
Purity: 100%
date received: 14/03/2012
Expire date: Indefinite
Storage Temperature: Room temperature

In vitro test system

Test system:

human skin model

Source species:

other: Reconstructed human model - in vitro

Cell type:

non-transformed keratinocytes

Cell source:

other: normal human keratinocytes cultured

Source strain:

other: Reconstructed human model - in vitro

Details on animal used as source of test system:

Not applicable

Justification for test system used:

SkinEthic is devoted to develop and produce reliable and robust in vitro alternative methods to animals.

Vehicle:

not specified

Details on test system:

Tissues were treated with the test item for 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hrs.
A volume of 10 µl of the test material was applied topically to the correspondent tissues ensuring uniform coverage. Triplicate tissues were treated with the test material for 15 minutes. In addition, triplicate tissues were exposed to DPBS (negative control) and 10 µl of SDS 5% w/v (positive control). After 7-minute contact time the SDS solution was re-added to maintain the distribution uniform for the remaining contact time.
The plates were stored at room temperature for 15 minutes.
At the end of the exposure time, each tissue was removed and rinsed and gently transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37 C, 5% CO2 in air for 42 hrs.
After 42 hrs of exposure, each 12 well plate was placed onto a place shaker for 15 minute to homogenise the released mediators in the medium. 1.6 ml of that medium was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°C for possible inflammatory mediator determination.
2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium was pipetted into the third column of 3 wells of 12 well plates. The tissues were transferred to the MTT filled wells and incubated for 3 hrs and 37°C, 5% CO2 in air.
The tissues were exposed to MTT solution and incubated for 3 hrs at 37°C, 5% CO2 in air. After the exposure time, each tissue was placed onto absorbent paper and a total biopsy of the epidermis was conducted using the Episkin biopsy punch.
The epidermis was carefully separated from the collagen matrix using forceps and both sides were placed into 1.5 ml microtubes containing 500µl of acidified isopropanol. Each tube was then mixed and refrigerated at 1 to 10 °C C for 3 days, allowing the extraction of formazan crystal out of MTT-loaded tissues.
For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96 well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as blanks. The optical density (OD) was measured at540 nm using Anthos 2001 microplate reader.

Control samples:

other: Dulbecco's Phosphate Buffered Saline (DPBS) was used as negative control. Sodium Dodecyl Sulphate (SDS) 5% W/v was used as the positive control

Test system

Duration of treatment / exposure:

Tissues were treated with the test item for 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hrs.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

88.8

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Time point: After 15-Minutes exposure period. (migrated information)

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

The test item Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers is not consider to be a skin irritant under the condition of this study.

Executive summary:

The study evaluated the irritation potential of the test item Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers using the Episkin Reconstructed human epidermis model after a treatment of 15 minutes followed by a post exposure incubation period of 42 hours. The study was performed according to the OECD 439 Guideline and under GLP conditions.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours and then taken for MTT loading.

The maintenance medium from each tissue was transferred to pre-labelled microtubes and stored in a freezer for possible inflammatory mediator determination. After MTT leading a total biopsy was conducted and each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystal out of MTT- loaded tissues.

At the end of the formazan extraction period each tube was mixed and duplicated 200 µl samples were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density was measured at 540 nm. The results showed that the relative mean viability of the test item treated tissues was 88.8% after the 15 -Minutes exposure period, therefore the test item was considered to be non-irritant.