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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2016 - 17 May 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
GLP study conducted according to OECD test Guideline No. 492 without any deviation.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
French GLP Compliance Programme for chemical products (inspected on 23 - 24 April 2015 / signed on 23 October 2015)

Test material

Constituent 1
Reference substance name:
alpha-pinene and beta-pinene dimers
alpha-pinene and beta-pinene dimers
Constituent 2
Reference substance name:
alpha-pinene and beta-pinene trimers
alpha-pinene and beta-pinene trimers
Constituent 3
Reference substance name:
alpha-pinene and beta-pinene tetramers
alpha-pinene and beta-pinene tetramers
Constituent 4
Reference substance name:
alpha-pinene and beta-pinene pentamers
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

Test animals / tissue source

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

- 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.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
other: Killled control tissue
Amount / concentration applied:
- 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

Results and discussion

In vitro

Irritation parameter:
other: % Tissue viability
mean corrected percent viability
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Visible damage on test system: Not specified


- 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

Any other information on results incl. tables

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




 0.906  0.776  1116.83 100.00      33.66        




0.645   83.17
Positive control  3




 0.313 0.292      40.36  37.65    5.42      UN GHS Category 2 or 1   




 0.271 34.95 
Test item      5




 0.678  0.664  87.427 85.62      3.61              




 0.650  83.817
Test item killed control      13





0.043      5.803  5.54     0.52   




 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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
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 (EpiOcularTMtissue 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.