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Description of key information

The skin and eye irritation potential of Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers have been evaluated. There was no evidence of skin and eye irritation or corrosion.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant and OECD/EU Method Guidelines. Validated in vitro test system, no restrictions
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)
Species:
other: Episkin Model Kit
Strain:
other: human epidermis model
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.
Details on study design:
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.

Irritation / corrosion parameter:
other:
Value:
88.8
Remarks on result:
other:
Remarks:
Time point: After 15-Minutes exposure period. (migrated information)
Irritant / corrosive response data:
Relative mean viability

Test item: 88.8% (sd: 9.8%)
Positive control: 7.4% (sd: 2.9%)
Negative control: 100% (sd 1.3%)
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
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.

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
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
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:
other: 24 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 48 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: Day 4, Day 7
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 48 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Time point:
other: Day 4, Day 7
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24 h
Score:
2
Max. score:
4
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 48 h
Score:
0.33
Max. score:
4
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: Day 4, Day 7
Score:
0
Max. score:
0
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.


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 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

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 corrosivity potential of 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 (Harlan Laboratories, 2012c). 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. No decrease in cell viability was apparent at either timepoint indicating that the test substance was not corrosive. The relative mean viability of the positive control item treated tissues (8N potassium hydroxide) 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 corrosive in vitro under the test conditions.

The second study (Harlan 2012d) investigated the irritation potential of the test item Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers using the EpiSkin Reconstructed human epidermis model. 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 viability assessed using MTT loading. 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 not irritating to skin.

A third study conducted by Pharmakon Research International (1981) investigated the potential of Terpenes and terpenoids, turpentine oil, alpha-pinene fraction oligomers to induce eye irritation in 9 New Zealand rabbits. The test material was instilled in the right eye of the rabbits. The treated eyes of 3 rabbits were rinsed for one minute with lukewarm water immediately following administration of the test article. The treated eyes of remaining 6 rabbits were unwashed. One positive ocular reading was recorded at 24 hrs post administration. Only one positive response (mild redness in one animal) was observed during the course of the study. Based upon the observation made in the Primary Eye Irritation test, the test material was not an eye irritant.

Justification for classification or non-classification

Based on the available studies and in accordance to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for skin and eye irritation.