Cyclohexanemethanol, 4-(1-methylethyl)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 August 2014 - 18 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted according to OECD Testing Guideline and was compliant per GLPs.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

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:

26 July 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)

Test material

Test material information

Constituent 1

Reference substance name:

cis-4-(isopropyl)cyclohexanemethanol

EC Number:

237-539-8

EC Name:

cis-4-(isopropyl)cyclohexanemethanol

Cas Number:

13828-37-0

Molecular formula:

C10H20O

IUPAC Name:

cis-(4-propan-2-ylcyclohexyl)methanol

Constituent 2

Reference substance name:

trans-(4-propan-2-ylcyclohexyl)methanol

Cas Number:

13674-19-6

Molecular formula:

C10H20O

IUPAC Name:

trans-(4-propan-2-ylcyclohexyl)methanol

Test material form:

liquid

Details on test material:

- Physical state: Clear colourless liquid
- Storage condition of test material: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation.

Vehicle:

unchanged (no vehicle)

Details on test system:

- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 14-EKIN-030
- Production date: not reported
- Shipping date: not reported
- Delivery date: 12 August 2014
- Expiry date: 18 August 2014
- Date of initiation of testing: 13 August 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: not reported. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without a reference filter)
- Filter: not appicable
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: no data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 0.847, 0.771 and 0.933
- Barrier function: IC50 = 2.1 mg/ml ( ≥ 1.5 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thinck stratum corneum
- Contamination: on the blood of the donor, absence of HIV1 and 2 antibodies, heptatitis C antobodies, hepatitis B antgen HB. On the epidermal cells absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): 10 μL (26.3 μL /cm2) of the test item was applied to the epidermis surface.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Lot/batch no 1528370

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Lot/batch no 1294323
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution

Duration of treatment / exposure:

The EpiSkin™ human epidermis skin constructs were treated with the undiluted test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 h.

Number of replicates:

Triplicate tissues for test item, negative and positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

15.3

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: yes

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

Any other information on results incl. tables

Direct MTT Reduction: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Table 7.3.1/1: Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.847	0.850	0.081	99.6	100*	9.6
	0.771			90.7
	0.993			109.8
Positive Control Item	0.218	0.208	0.011	25.6	24.5	1.3
	0.196			23.1
	0.211			24.8
Test Item	0.125	0.130	0.008	14.7	15.3	1.0
	0.126			14.8
	0.139			16.4

Assay validity:

- The relative mean tissue viability for the positive control treated tissues was 24.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.3%. The positive control acceptance criterion was therefore satisfied.

- The mean optical density for the negative control treated tissues was 0.850 and the standard deviation value of the percentage viability was 9.6%. The negative control acceptance criterion was therefore satisfied.

- The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 1.0%. Therefore, the test item acceptance criterion was satisfied.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant)

Conclusions:

Under the experimental conditions of this study, the test item is classifided as irritant to the skin (Category 2) according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item. Triplicate tissues were treated with 10 μL (26.3 μL/cm2) of the undiluted test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5‑diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

This assay was valid with negative and positive controls showing results within the acceptable range.

The test substance with a mean tissue viability of 15.3 ± 1.0 %, was predicted as irritant to the skin. The quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test item is classifided as irritant to the skin (Category 2) according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.