(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; Identification of chemicals not requiring classification and labeling for eye irritation or serious eye damage

Version / remarks:

2021-02-02

Deviations:

no

Test guideline 2

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:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

EC Number:

225-591-4

EC Name:

(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

Cas Number:

4948-28-1

Molecular formula:

C10H18O

IUPAC Name:

(1R,2S,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-2-ol

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µg

Duration of treatment / exposure:

6 hours at 37 ± 1.5°C

Duration of post- treatment incubation (in vitro):

25 min at room temperature (post-exposure immersion) + 18 hours at 37 ± 1.5°C (post-exposure incubation)

Number of animals or in vitro replicates:

2 tissue replicates per test item and control treatment

Details on study design:

- Details of the test procedure used
- Doses of test chemical and control substances used: Test and control substances were tested neat
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 hours at 37 ± 1.5°C (exposure), 25 min at room temperature (post-exposure immersion) + 18 hours at 37 ± 1.5°C (post-exposure incubation)
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
Colouring test chemicals: Two additional controls in duplicates run with the main experiment (additional viable tissues (colored controls = CC)) were used. Two tissues were treated with deionised water (NC_CC) and two tissues were treated with the test item (TI_CC). These four tissues were incubated in medium without MTT solution in the MTT assay. At the end, Data Correction Procedure I was performed.
- Number of tissue replicates used per test chemical and controls: 2 tissues (positive control, negative control, NSCliving)
- Wavelength used for quantifying MTT formazan: 570 nm (Versamax® Molecular Devices)
- Description of the method used to quantify MTT formazan: The optical density (OD570nm) was determined spectrophotometrically in duplicates by a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: See "Any other information on materials and methods incl. tables"
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: See "Attached background material"
- Complete supporting information for the specific RhCE tissue construct or hCE cells used: See "Attached background material"
- Reference to historical data of the RhCE tissue construct: A Certificate of Analysis was provided by the supplier showing that all specifications were within the acceptance criteria defined by the supplier.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The technical proficiency of the test system according to OECD Guideline 492 guideline recommended proficiency substances was demonstrated. A Certificate of Proficiency is available.
- Positive and negative control means and acceptance ranges based on historical data: Acceptance ranges were met, see "Attached background material
- Acceptable variability between tissue replicates for positive and negative controls: Acceptance ranges were met, see "Attached background material
- Acceptable variability between tissue replicates for the test chemical: Acceptance ranges were met, see "Attached background material

Results and discussion

In vitro

Results

Irritation parameter:

percent tissue viability 

Run / experiment:

Mean of two indipendent runs

Value:

1.21

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the test system according to OECD Guideline 492 guideline recommended proficiency substances was demonstrated. A Certificate of Proficiency is available.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values: See "Attached background material"

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

Conclusions:

In an in vitro eye irritation study according to OECD guideline 492, the test item was either irritating or corrosive to eyes. As no prediction can be made for the test item from this result in isolation, additional information for classification purposes is required.

Executive summary:

This in vitro study was performed according to OECD guideline 492 and GLP to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Therefore, additional tests with freeze-killed tissues did not have to be performed. The test item proved to dye water in the color interference pre-experiment since the OD of the test item in deionised water at 570 nm after blank correction was > 0.08. Therefore, additional tests with viable tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the main experiment. Each 50 mg of the test item or 50 μL of the negative control (deionised water) and of the positive control (methyl acetate), were applied to each duplicate tissue for 6 hours. The mean OD of the tissue replicates treated with the negative control was > 0.8 and < 2.8, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of relative viability between the two relating tissues was < 20 p.p. in the same run (for test item tissues, positive and negative control tissues). After treatment with the test item a mean relative viability value of 1.21% was measured compared to the mean value of the negative control. This value is below the threshold for irritancy of ≤ 60%. In conclusion, it can be stated that in this study and under the experimental conditions reported, no prediction can be made for the test item from this result in isolation and requires additional information for classification purposes.