Oligomerisation products of alpha-pinene and beta-pinene

Endpoint summary

Description of key information

According to a GLP study conducted in accordance with OECD Guideline 439, the test item is not irritant to skin.

Acoording to a GLP study conducted in accordance with OECD Guideline 492, no classification is required for eye irritation or serious eye damage.

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:

30 November 2016 - TBC

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 439 and in compliance with GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

French GLP Compliance Programme for chemical products (inspected on 23 - 24 April 2015 / signed on 23 October 2015)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 16-RHE-130
- Production date: Not reported
- Shipping date: Not specified
- Delivery date: 13 December 2016
- Expiry date: 19 December 2016
- Date of initiation of testing: 13 December 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 x 1 mL of DPBS (PAN BIOTECH GmbH, Batch No. 9510916)
- Observable damage in the tissue due to washing: The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues.
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek
- Wavelength: 570 nm
- Filter: Not applicable
- Filter bandwidth: Not applicable
- Linear OD range of spectrophotometer: No data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570= 1.5, CV= 7.8% (specification: OC > 0.7)
- Barrier function: ET50 (' Exposure time inducing 50% viability using Triton X-100 1%)= 4.7h (specification: 4.0h =< ET50 =< 10.0h)
- Morphology: well differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: : absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- 2 killed control tissue models were added to the study due to the direct reduction of MTT (non-specific MTT reduction control) (Episkin SA, RHE/S/17 Batch No. 16-RHE-098, frozen on 13 September 2016).
- No need to add non-specific coloration control since the test item has no coloration potential.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes of exposure and 42 hours of post-treatment incubation is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL of the test item was then applied to the epidermal surface.
- Concentration (if solution): not applicable

VEHICLE
not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of DPBS
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of SDS
- Concentration (if solution): 5% w/v aqueous solution

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 42 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 41 hours and 8 minutes in fresh medium.

Number of replicates:

3 living tissues for test item, negative and positive controls and 2 killed tissues (for non-specific MTT reduction control)

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean corrected percent viability

Run / experiment:

1

Value:

59.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

1.5%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

__TBC__
OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: Yes
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: No, the standard deviation value which was 26.0% instead of lower than or equal to 18% initially scheduled. This was due to an optical density measured for epidermises No.1 and No.2 slightly above the range of historical data. Considering the results obtained, this deviation is considered as without impact on the conclusion of the study.
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: TBC

Table 7.3.1/1: Mean OD₅₇₀Values and Percentage Viabilities for the Negative Control Item, Positive Control Item, Test Item Killed Tissue and Test Item

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD	Conclusion
Negative control	1	2.015 1.908 1.969	1.964	1.574	124.8	100.0	26.0
	2	1.513 1.632 1.685	1.610		102.3
	3	1.063 1.190 1.188	1.147		72.9
Positive control	4	0.027 0.028 0.027	0.027	0.023	1.7	1.5	0.5	Irritant
	5	0.015 0.015 0.015	0.015		1.0
	6	0.027 0.029 0.028	0.028		1.8
Test item	13	1.048 1.040 1.046	1.045	0.983	66.4	62.5	15.0
	14	1.201 1.188 1.158	1.182		75.1
	15	0.743 0.713 0.713	0.723		45.9
Test item killed tissues	25	0.052 0.053 0.049	0.051	0.048	3.2	3.1	0.3
	26	0.045 0.046 0.044	0.045		2.9
Test item corrected						59.4		Non irritant

#: mean of 3 values (triplicate of the same extract)

OD: optical density

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

Acceptability criteria: SD ≤ 18%.

Note: The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

Interpretation of results:

GHS criteria not met

Conclusions:

Test item TERPENIC OLIGOMERS has to be considered as non-irritant to skin according to CLP regulation (EC) No.1272/2008. It is also not classified according to UN GHS Regulation.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439, the EU Method B.46 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model.

Test item TERPENIC OLIGOMERS was applied as supplied at the dose of 16 μL, to 3 living and 2 killed Reconstructed Human epidermis (SkinEthic RHE® model) during 43 minutes, followed by a rinse with 25 mL of DPBS and a 41-hour and 8-minute post-incubation period at 37°C, 5% CO2.

Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

 

The mean corrected percent viability of the treated tissues was 59.4%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate).

 

Therefore, test item TERPENIC OLIGOMERS has to be considered as non-irritant to skin according to CLP regulation (EC) No. 1272/2008. It is also not classified according to UN GHS Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 November 2016 - 17 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 492 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 28 July 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

French GLP Compliance Programme for chemical products (inspected on 23 - 24 April 2015 / signed on 23 October 2015)

Species:

other: Reconstructed human cornea-like epithelium tissues (EpiOcular™ tissue model)

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. This test guideline is applicable to liquids and semi-solids, so is considered to be applicable to the test item.

CELL SYSTEM:
- EpiOcular™ OCL-212-ver2.0, supplied by MatTek Corporation (Bratislava, Slovakia).
- Lot No.: 23755
- Keratinocyte strain: 4F1188
- Analysis for potential biological contaminants: none detected (HIV-1, Hep. B and Hep. C virus; Bacteria, yeast and other fungi)
- Tissue viability: 1.355, within the acceptance criteria (1.1-3.0)
- Barrier function: 16.42 min, within the acceptance criteria (12.2-37.5)
- Sterility: Sterile
- Transport: Not specified
- Storage: On day of receipt of the EpiOcular™ tissues, the 12-tissues in their 24-well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium (MatTek Corporation, batch No. 120516MG) and incubated during 19 hours and 58 minutes at standard culture conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: Killled control tissue

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): not applicable

VEHICLE: Not applicable

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

- 12-minute immersion period at room temperature
- 2-hour incubation at 37°C, 5% CO2

Number of animals or in vitro replicates:

2 replicates

Details on study design:

- Details of the test procedure used : procedure for liquids
* Pre-treatment: after an overnight incubation, tissues were pre-wetted with 20 µL of Ca2+Mg2+Free-DPBS. Then tissues were incubated for 30 minutes at standard culture conditions.
* Treatment: 50 µL of test item, positive or negative control was applied to the entire surface of 2 living RhCE tissue replicates and 2 killled DPBS pre-treated RhCE tissue replicates during 30 minutes at standard culture conditions.
* Post-exposure incubation period: after treatment, tissues were carefully washed by extensive rinsing with Ca2+Mg2+Free-DPBS. Rinsed tissues were checked for any coloration and for comparable colour with negative control treated tissues (whitish). The rinsing step was followed by a 12-minute immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. Then the RhCE constructs were incubated for 2 hours at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- Doses of test chemical and control substances used : 50 µL

- Duration and temperature of pre-treatment (30 minutes), exposure (30 minutes), post-exposure immersion (2 hours)

- Description of any modifications to the test procedure : None

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : Since the test item was identified as a direct MTT reducer, two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non­specific MTT reduction control. The test item did not interfere with the MTT assay, thus no non-specific coloration control was added to the study.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving): 2

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is identified potentially requiring classification and labelling according to UN GHS (Category 2 of Category 1.
According to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Yes, attached to the study report.
* Historical negative control ranges 83.17 - 116.83%
* Historical positive control ranges 6.50 - 60.03%

- Complete supporting information for the specific RhCE tissue construct used : Yes, attached to the study report.

- Reference to historical data of the RhCE tissue construct : No

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Yes, attached to the study report.

- Positive and negative control means and acceptance ranges based on historical data : Yes

- Acceptable variability between tissue replicates for positive and negative controls: Yes

- Acceptable variability between tissue replicates for the test chemical: Yes

Irritation parameter:

other: % Tissue viability

Remarks:

mean corrected percent viability

Run / experiment:

1

Value:

80.08

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.00%

Positive controls validity:

valid

Remarks:

37.65%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Met, except for the difference of viability between the two tissues of the negative control group which was 33.66%, instead of =< 20% as initially scheduled. Considering the results obtained, this deviation has no impact on the conclusion of the study.
- Acceptance criteria met for positive control: met

Table 7.3.2/1: Mean OD₅₇₀Values and Percentage Viabilities for the Negative Control Item, Positive Control Item, Test Item Killed Tissue and Test Item

	Tissue	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Difference of viability %	Conclusion
Negative control	1	0.911 0.892 0.917	0.906	0.776	1116.83	100.00	33.66
	2	0.617 0.659 0.660	0.645		83.17
Positive control	3	0.308 0.313 0.319	0.313	0.292	40.36	37.65	5.42	UN GHS Category 2 or 1
	4	0.269 0.273 0.271	0.271		34.95
Test item	5	0.666 0.679 0.689	0.678	0.664	87.427	85.62	3.61
	6	0.658 0.654 0.638	0.650		83.817
Test item killed control	13	0.043 0.048 0.045	0.045	0.043	5.803	5.54	0.52
	14	0.036 0.044 0.043	0.041		5.287
Test item corrected						80.08		No Category

Note: the optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

 

 

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Interpretation of results:

GHS criteria not met

Conclusions:

Test item TERPENIC OLIGOMERS does not require classification for eye irritation or serious eye damage according to CLP Regulation EC No. 1272/2008 and GHS regulations.

Executive summary:

A study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The study was conducted according to OECD Guideline No. 492 and in compliance with GLP.

Test item TERPENIC OLIGOMERS was applied, as supplied, at the dose of 50 µL, to 2 living and 2 killed PBS pre-treated RhCE (EpiOcular^TMtissue model) during 30 minutes at 37°C, 5% CO₂, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 12-minute post-exposure immersion period at room temperature and a 2-hour post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

The mean corrected percent tissue viability of the RhCE replicates treated with test item TERPENIC OLIGOMERS was 80.08%, versus 37.65% in the positive control (Methyl acetate).

Therefore, test item TERPENIC OLIGOMERS does not require classification for eye irritation or serious eye damage according to CLP Regulation EC No. 1272/2008 and GHS Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

A key study (Colas, 2018, Rel.1) was performed to evaluate the skin irritation potential of the test substance, Terpenic oligomers. This in vitro skin irritation study was performed according to OECD Guideline 439 and in compliance with GLP, using the SkinEthic Reconstructed Human Epidermis® model.

Test item TERPENIC OLIGOMERS was applied as supplied at the dose of 16 μL, to 3 living and 2 killed Reconstructed Human epidermis (SkinEthic RHE® model) during 43 minutes, followed by a rinse with 25 mL of DPBS and a 41-hour and 8-minute post-incubation period at 37°C, 5% CO2.

Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The mean corrected percent viability of the treated tissues after 42-minute exposure was 59.4%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate).

Therefore, test item TERPENIC OLIGOMERS has to be considered as non-irritant to skin according to CLP regulation (EC) No. 1272/2008. It is also not classified according to UN GHS Regulation.

Eye irritation:

A key study was identified for eye irritation. This in vitro eye irritation test (Colas, 2017, Rel.1) was performed according to OECD Guideline 492 and in compliance with GLP, using the Reconstructed human Cornea-like Epithelium (RhCE). The test item, terpenic oligomers, was applied as supplied at the dose of 50 µL, to 2 living and 2 killed DPBS pre-treated RhCE (EpiOcularTM tissue model) during 30 minutes at 37°C. The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 -minute post-exposure immersion period at room temperature and a 2-hour post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay. The mean corrected percent tissue viability of the RhCE replicates treated with test itemTERPENIC OLIGOMERS was 80.08%, versus 37.65% in the positive control (Methyl acetate).

Therefore, test item TERPENIC OLIGOMERS does not require classification for eye irritation or serious eye damage according to CLP Regulation EC No. 1272/2008 and the UN GHS Regulation.

Respiratory irritation:

No data was available regarding respiratory irritation assessment.

Justification for classification or non-classification

Harmonized classification:

Test item terpenic oligomers does not have harmonized classification according to Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, the substance should not be classified as skin irritant according to CLP Regulation (EC) No. 1272/2008 and to the UN GHS Regulation.

Based on the available information, the substance should not be classified as eye irritant according to CLP Regulation (EC) No. 1272/2008 and to the UN GHS Regulation.

No data was available regarding respiratory irritation. However, the substance not being skin or eye irritant, no classification is expected for respiratory irritation.