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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-02-15

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:

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

Version / remarks:

2021-06-14

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:

2012-07-06

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2020-04-12

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200)

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcularTM 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

Remarks:

The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.

Principles of method if other than guideline:

The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Reconstructed Human EpiDerm model purchased from MatTek Corporation (82105 Bratislava, Slovakia)
- Tissue batch number: 36101
- Date of certificate of analysis: 2021-10-27
- Date of initiation of testing: 2021-10-12

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5°C
- Temperature of post-treatment incubation: 24 ± 2 hours at room temperature and further 18 ± 2 hours at 37 ± 1.5°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were gently rinsed with PBS several times
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours ± 5 minutes
- Spectrophotometer: Versamax® (Molecular Devices)
- Wavelength: OD 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was checked for the tissue batch by the supplier and was found to be within the acceptable range. Concurrent negative controls (NC) were used in each run to demonstrate that viability (with the NC) of the tissues were within a defined historical acceptance range (see attached background material).
- Barrier function: Barrier function was checked for the tissue batch by the supplier and was found to be within the acceptable range. Concurrent positive controls (PC) were used in each run to demonstrate that barrier function and resulting tissue sensitivity of the tissues were within a defined historical acceptance range (see attached background material).
- Contamination: Every tissue batch was checked for biological contaminants. No HIV-1 virus, Hepatitis B or C virus and no bacteria, yeast and other funi were detected.
- Reproducibility: All acceptance criteria were met (for the tissue batch and the tissue replicates used in the study), thus, reproducibility was proven.

NUMBER OF REPLICATE TISSUES: 3 tissue replicates per test item/positive and negative control treatment

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was > 0.08 in the first pre-experiment, it was presumed to have the potential to stain the tissue.
- Fresh tissues were used to control for color interference
- N. of replicates: Two controls in duplicates run (2 deionised water treated tissues and 2 test item treated tissues)
- Method of calculation used: Data Correction Procedure I was performed (see "Any other information on materials and methods incl. tables")


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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating or corrosive to skin if the viability after 1 hour exposure and 42 hours post-incubation is equal or less than 50%.
- The test substance is considered to be non-irritating to skin if the viability after 1 hour exposure and 42 hours post-incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 ± 2 mg (39.7 mg/cm2 according to guideline)

Duration of treatment / exposure:

60 minutes (35 minutes at 37 ± 1.5°C and 5 ± 0.5% CO2 and 25 min at room temperature)

Duration of post-treatment incubation (if applicable):

42 ± 4 hours

Number of replicates:

The test item and the controls, respectively, were tested in triplicate tissues

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 3 tissue replicates

Run / experiment:

1

Value:

6.9

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
- Direct-MTT reduction: No
- Colour interference with MTT: Yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of EpiDermTM SIT for regulatory purposes, technical proficiency was demonstrated by correctly predicting the proficiency chemicals listed in OECD TG 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: 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 skin irritation study according to OECD guideline 439, the test item was identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, since the tissue viability after exposure and post-incubation was less or equal to 50%.

Executive summary:

This in vitro study according to OECD guideline 439 and GLP was performed to assess the skin irritation potential of the test item by means of the Human Skin 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. Three tissues each of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. After treatment with the test item, the mean relative viability value was 6.90% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin. The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, since the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.