2,6-dimethyloct-7-en-2-ol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-08-03 to 2021-12-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

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 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2021-06-14

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,6-dimethyloct-7-en-2-ol

EC Number:

242-362-4

EC Name:

2,6-dimethyloct-7-en-2-ol

Cas Number:

18479-58-8

Molecular formula:

C10H20O

IUPAC Name:

2,6-dimethyloct-7-en-2-ol

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: Adult human donors

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The EPISKIN model has been validated for irritation testing 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. (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm SIT (MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue batch number: 34190
- Date of analysis of lot tissue functionality and quality: 2021-09-01

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: At 37°C ± 1.5°C and 5 ± 0.5% CO2 for 35 minutes and afterwards at room temperature for 25 minutes

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The units were gently rinsed with PBS several times. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of 1 mg/mL MTT per well
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability, barrier function and reproducibility: The quality of the final product is assessed via:
1) MTT cell viability test (n=3), acceptance criteria: OD (540-570) [1.0-3.0) (for tissue viability)
2) ET-50 assay using 1% Trition X-100 (n=3) and MTT assay, acceptance criteria: ET50 [4.77-8.72 hours) (barrier function)
3) Long-term antibiotic and antimycotic free culture, acceptance criteria: no contamination (sterility)
- Contamination: The absence of HIV1 and 2 antibodies, Hepatitis C antibodies and Hepatitis B antigens HBs as well as funghi, bacteria and mycroplasma were verified beforehand.

NUMBER OF REPLICATE TISSUES: 3 replicate tissues per test item, negative and positive control. Duplicate measurement per replicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues: Since no non-specific colouring potential of the test item was detected in a pre-experiment, no fresh tissue controls were used.
- Killed tissues: The test item did not show direct interaction with MTT. The use of additional killed tissue controls was not necessary.
- N. of replicates: 3 replicates per test item, 3 replicates per controls (negative, positive controls) were used. No colour or MTT interference controls were used.
- Method of calculation used: 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 irritant or corrosive to skin (category 2 or 1) if the viability after 60 min exposure is equal or less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 min exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 μL

NEGATIVE CONTROL
- Amount applied: 30 μL

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5% SDS in deionized water

Duration of treatment / exposure:

60 minutes (35 minutes at standard incubation conditions and 25 minutes at room temperature).

Duration of post-treatment incubation (if applicable):

42 ± 4 hours (in 0.9 mL of assay medium for 24 ± 2 hours and, after a medium change, another 18 ± 2 hours) at standard incubation conditions.

Number of replicates:

3 replicate tissues per test item, negative and positive control were tested in a single independent experiment.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 (mean of 3 tissue replicates)

Value:

3.14

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: No

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:

This test method cannot resolve between UN GHS Categories 1 and 2, thus classification is only possible in combination with OECD guideline 431.

Conclusions:

In an in vitro skin irritation study according to OECD guideline 439, the test item is irritant to skin according to UN GHS and EU CLP regulation and under the experimental conditions reported.

Executive summary:

This in vitro study according to OECD guideline 439 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. Also, its intrinsic color was not intensive and the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. 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 3.14% 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.