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Administrative data

Description of key information

Information on the skin irritation and corrosion potential of Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers is available from a GLP compliant, OECD guideline studies conducted by Harlan Laboratories Ltd (2012c,d).

The first 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.

A second study evaluated the corrosivity potential of the test item Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers using the Skinethic in vitro Reconstructed Human Epidermal (RHE) Model after 3 minutes and 60 minutes of treatment.

The study was performed according to the OECD 431 Guideline and under GLP conditions.

Cytotoxicity was determined by the metabolic conversion of the vital dye MTT formazan by viable cells in the test item treated cultures relative to the negative control. The results were obtained calculating the relative mean viability from absorbance measurements. The result showed that the MTT solution containing the test material di not turn blue which indicated that the test item did not directly reduce MTT. The relative mean viability of the positive control item treated tissues was 12.6% after the 3 -Minute exposure period and 2.8% after the 60 -Minute exposure period. In conclusion, the test item Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers was not considered to be corrosive in vitro under the test conditions.

Regarding the eye irritation assessment, the capability of Terpenes and terpenoids, turpentine oil, alpha-pinene fraction oligomers to induce eye irritation was evaluated using 9 New Zealand rabbits. The study design used is similar to the OECD 405 and it was performed under GLP conditions. The test material was instilled in the right eye of the rabbits. The treated eyes of 6 rabbits remained unwashed. The treated eyes of 3 rabbits were rinsed for one minute with lukewarm water immediately following administration of the test article. One positive ocular reading was recorded at 24 hrs post administration. Only one positive response was observed during the course of the study. Based upon the observation made in the Primary Eye Irritation test, the test material is not considered an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)
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
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
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.
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
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Remarks:
In vivo study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test Material: Zonarez A-25
Description: PC 12-67
Lot Number: AIA804
Special Instructions: Standard Precautions
Stability: There was no apparent change in the physical state of the test article during administration
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Perfection Breeders, Inc.
- Weight at study initiation: 2-3 kg
- Fasting period before study:
- Housing: Separate isolation by test system
- Diet (e.g. ad libitum): Wayne Rabbit Ration, ad libitum, checked daily and added or replaced as needed.
- Food Analysis: Acute dose minimize the effect of contaminants. There were no contaminants.
- Water (e.g. ad libitum): Fresh tap water, fit for human consumption, ad libitum, using 16 ounce glass bottles with rubber stopper and stainless steel sipper tube or an automatic watering system supplied by Edstrom Industries Inc.
- Water Analyses: Conducted by Pennsylvania Gas and Water Company
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C+/-3°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hrs light-12 hrs dark
Vehicle:
not specified
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Dose administration: 0.1 ml, undiluted

VEHICLE
- Information not reported
Duration of treatment / exposure:
Once
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
9 rabbits, 6 treated-no rinsed, 3 treated-rinsed
Details on study design:
Methods of the study performance
Twenty-four hrs prior to the application of the test material, the eyes of the rabbit were examined using 2% sodium fluorescein stain. Animals with preexisting corneal injury were removed from the study. The test substance was placed in the right eye of each animal by gently pulling the lower lid away from the eyeball to form a cup. The upper and lower lids were then gently held together for one second to prevent loss of material.
The treated eyes of six rabbits remained unwashed. The treated eyed of three rabbits were rinsed for one minute with lukewarm water immediately following administration of the test material. The contralateral eye remained untreated as served as a control.

The eye were examined at 24, 48, 72 hrs and 4 and 7 days after treatment.
Grading of irritation is according to the method of Draize, J.H, et al., Pharm, Exp. Ther.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
48 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
7 d
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Remarks:
Day 4 and 7
Irritation parameter:
iris score
Basis:
mean
Time point:
24 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
48 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
7 d
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Remarks:
Day 4 and 7
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24 h
Score:
2
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
48 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
7 d
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Remarks:
Day 4 and 7
Irritant / corrosive response data:
One positive ocular reading was observed at 24 hrs post administration. No other positive responses were observed. One positive response were recorded during the course of the study.


Other effects:
No other effects were observed
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material Terpenes and terpenoids, turpentine oil, alpha-pinene fraction oligomers was not an eye irritant.
Executive summary:

The study investigated the capability of Terpenes and terpenoids, turpentine oil, alpha-pinene fraction oligomers to induce eye irritation in 9 New Zealand rabbits.

The study design used is similar to the OECD 405 and it was performed under GLP conditions.

The test material was instilled in the right eye of the rabbits. The treated eyes of 6 rabbits remained unwashed. The treated eyes of 3 rabbits were rinsed for one minute with lukewarm water immediately following administration of the test article. One positive ocular reading was recorded at 24 hrs post administration. Only one positive response was observed during the course of the study. Based upon the observation made in the Primary Eye Irritation test, the test material is not considered an eye irritant.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Not classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 orUN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).