rel-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 April 2016 - 27 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Updated guideline adopted July 28, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol “In vitro EpiDerm(TM) Skin Corrosion Test (EPI-200-SCT)”

Version / remarks:

07 November 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 14 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

rel-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]hexan-3-ol

EC Number:

814-113-5

Cas Number:

253454-23-8

Molecular formula:

C15H30O

IUPAC Name:

rel-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Test material form:

liquid

Specific details on test material used for the study:

Hysandol called Timberol in the test report may be a multi of the cis and trans-isomer, while Hysandol is a mono-trans isomer. The cis isomer is expected to have the same results for this endpoint because it is stereo isomer.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation (Bratislava, Slovakia)

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissue
- Tissue batch number(s): 23338
- Delivery date: 24 May 2016
- Date of initiation of testing: 24 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes exposure: at room temperature, 60 minute exposure: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times using a wash bottle containing DPBS
- Observable damage in the tissue due to washing: no

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
The substance was checked for possible interference with the MTT endpoint before the start of the study. 50 μL of the test item was added to 0.3 mL of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. The test item did not dye water when mixed with it.
To test if an item directly reduces MTT, 50 μL of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. The test item did not prove to be a MTT reducer.

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

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 exposure times

EVALUATION
The mean OD of the duplicate negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%)= [mean OD(test item/positive control)/mean ODnegative control] *100

PREDICTION MODEL / DECISION CRITERIA: see Table 1

ACCEPTABILITY CRITERIA:
1. The mean OD of the tissue replicates treated with the negative control should be ≥ 0.8 and ≤ 2.8 for every exposure time
2. The mean viability of the tissue replicates treated with the positive control for 1 hour, should be <15% compared to the negative control
3. The Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates should be ≤ 30%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL (79.4 μL/cm^2)

Duration of treatment / exposure:

3 +/- 0.5 minutes and 60 +/- 0.5 minutes

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure / mean of 2 replicates

Value:

97.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

16.2

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure / mean of 2 replicates

Value:

105.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

10.7

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.627 and 1.755)
- Acceptance criteria met for positive control: yes, the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% (10.7%) compared to the negative control
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (values between 0.2% and 5.8%)

Any other information on results incl. tables

Mean OD₅₇₀ values and viabilities for the negative control, positive control and test item are given below:

Item	Exposure Period (minutes)	Individual OD570 of Individual tissues	Mean OD570 of duplicate tissues (tvt)	Water-Killed Tissues			True Viability tvt-(tkt-ukt)	Relative mean viability (%)
				tkt	ukt	tkt-ukt
Negative Control Item	60	1.634	0.818					100*
		1.639
Positive Control Item		0.219	0.027					10.7
		0.186
Test Item	3	1.587	1.611					97.7
		1.635
	60	1.780	1.710					105.4
		1.640

*=   The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive to the skin

Remarks:

In accordance with EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

The results of an in vitro skin corrosion test showed that the substance was not corrosive to the skin (tissue viability after 3 minutes exposure: 97.7% and tissue viability after 60 minutes exposure: 105.4%).

Executive summary:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to the substance, a negative control (deionised water) and a positive control (8.0 N KOH) for 3 minutes and 60 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met. The cell viability of the tissues exposed to the substance were 97.4% and 105.4% for 3 minutes and 60 minutes exposure, respectively. Both values did not exceed thereshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), therefore the substance is considered not to be corrosive.