[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Apr - 22 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

EC Number:

216-365-6

EC Name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Cas Number:

1565-76-0

Molecular formula:

C11H22O

IUPAC Name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200-SIT Kit
- Tissue batch number: 21671
- Production date: not specified
- Shipping date: not specified
- Delivery date: 19 May 2015
- Date of initiation of testing: 19 May 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C for 35 min and room temperature for 25 minutes
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material, after the rinsing the inserts were submerged in DPBS at least three times.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Wavelength: 570 ± 1 nm
- Filter: 570 ± 1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 2.57 ± 0.089 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.96 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues was not performed.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 60 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30µL
- Concentration: 100%

NEGATIVE CONTROL
- Amount applied: 30 µL
- Concentration: 100%

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5%

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

40.5 hours

Number of replicates:

triplicates for each treatment and control group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min / test material

Value:

114.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes: After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: yes: Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.1%.
- Acceptance criteria met for variability between replicate measurements: yes: The relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 11% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%)

Any other information on results incl. tables

Table 2. Results after 60 min incubation time.

Test group	Absorbance at 570 nm			Mean absorbance	Rel. absorbance (%) Tissue 1, 2 and 3	Rel. SD (%)	Mean rel. absorbance (% of negative control)
	Tissue 1*	Tissue 2*	Tissue 3*
Negative control	1.993	2.064	2.064	2.040	97.7 101.1 101.1	2.0	100.0
Positive control	0.063	0.069	0.056	0.063	3.1 3.4 2.7	10.6	3.1
Test substance	2.118	2.390	2.484	2.331	103.8 117.2 121.7	8.2	114.2

* Mean of 3 replicate wells after blank correction

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test item/positive control) / (mean absorbance negative control)

The optical pre-experiment (colour interference pre-experiment) to investigate the test substance’s colour change potential in water did not led to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test substance after 1 h incubation with MTT-reagent did not show blue colour.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the Reconstructed Human Epidermis test the test substance does not possess any skin irritating potential.