Oligomerisation products of alpha-pinene and beta-pinene

Skin irritation / corrosion

Administrative data

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.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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)

Version / remarks:

Adopted 28 July 2015

Deviations:

no

Test guideline 2

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 material

Test material information

Constituent 1

Reference substance name:

alpha-pinene and beta-pinene dimers

IUPAC Name:

alpha-pinene and beta-pinene dimers

Constituent 2

Reference substance name:

alpha-pinene and beta-pinene trimers

IUPAC Name:

alpha-pinene and beta-pinene trimers

Constituent 3

Reference substance name:

alpha-pinene and beta-pinene tetramers

IUPAC Name:

alpha-pinene and beta-pinene tetramers

Constituent 4

Reference substance name:

alpha-pinene and beta-pinene pentamers

IUPAC Name:

alpha-pinene and beta-pinene pentamers

Test material form:

liquid: viscous

Details on test material:

Batch No. 16-03 SI
Purity: 100% (UVCB substance)
Name of the test item (as cited in the study report): TERPENIC OLIGOMERS
Physical state: amber viscous liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Reassay date: 05 January 2017

In vitro test system

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)

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.