4-tert-butylcyclohexyl acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2012-09-04 to 2012-10-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 439 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

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:

22 July 2010

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:

2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Monitoring Programme (inspected on 10 July 2012)

Test material

Test material information

Constituent 1

Reference substance name:

trans-4-tert-butylcyclohexyl acetate

EC Number:

217-598-6

EC Name:

trans-4-tert-butylcyclohexyl acetate

Cas Number:

1900-69-2

Molecular formula:

C12H22O2

IUPAC Name:

trans-4-tert-butylcyclohexyl acetate

Constituent 2

Reference substance name:

cis-4-tert-butylcyclohexyl acetate

EC Number:

233-881-7

EC Name:

cis-4-tert-butylcyclohexyl acetate

Cas Number:

10411-92-4

Molecular formula:

C12H22O2

IUPAC Name:

cis-4-tert-butylcyclohexyl acetate

Test material form:

liquid

Details on test material:

Description: Clear colorless liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Several in vivo studies were presumably conducted on the substance. However discordant results were reported, bringing doubts on the identity, purity and composition of the tested substances. As it was not possible to obtain certificates of analysis of the tested batches, it was deemed necessary to conduct a new in vitro study on the substance.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Supplier: SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 12-EKIN-037
- Production date: not reported
- Shipping date: not reported
- Delivery date: 09 October 2012
- Date of initiation of testing: 09 October 2012 (Pre-incubation); 10 October 2012 (main test)
- Expiry date: 15 October 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature, 15 minutes
- Temperature of post-treatment incubation (if applicable): 37°C, 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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.
- Observable damage in the tissue due to washing: none observed
- Modifications to validated SOP: none

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: 540 nm
- Filter: without a reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 22.2 ± 0.3 (CV = 1.2 %) (≥ 19.5)
- Barrier function: IC50 = 2.1 mg/ml (IC50 ≥ 1.5 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination:
On blood : absence of HIV1 and 2 antibodies (Architect Abbott); absence of hepatitis C antibodies (Architect Abbott); absence of hepatitis B antigen HBs (Architect Abbott).
On epidermal cells: absence of bacteria, fungus and mycoplasma.
- Reproducibility: All values for the control groups were within the ranges obtained by the testing laboratory in the preceding 120 studies. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: Triplicate tissues for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable, the test item did not directly reduce MTT.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Relative mean tissue viability is ≤50%: Irritant
- Relative mean tissue viability is >50%: Non-Irritant (CLP)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): as supplied.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Test system

Vehicle:

unchanged (no vehicle)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

62.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

5.8%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean OD540 for the negative control treated tissues was 0.740 and the standard deviation value of the viability was 6.62%. The negative control
acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: Yes; the relative mean tissue viability for the positive control treated tissues was 5.8% relative to the negative control treated tissues and the standard devi
ation value of the viability was 0.7%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.9%. The test acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: All values for the control groups were within the ranges obtained by the testing laboratory in the preceding 120 studies.

Any other information on results incl. tables

Table 7.3.1/1: Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD540 of tissues	Mean OD540 of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.684	0.740	0.049	92.4	100*	6.6
	0.762			103.0
	0.773			104.5
Positive Control Item	0.044	0.043	0.005	5.9	5.8	0.7
	0.038			5.1
	0.048			6.5
Test Item	0.437	0.463	0.059	59.1	62.6	6.9
	0.431			58.2
	0.522			70.5

SD=   Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test material is not classified as irritating to skin in this in vitro assay according to the criteria of the Regulation (EC) No (1272/2008 (CLP).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

This test was designed to be compatible with the OECD Guideline No. 439 and was performed in compliance with GLP.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre‑labelled 96‑well plate. The optical density was measured at 540 nm.

The relative mean viability of the test item treated tissues was 62.6 ± 6.9 %, after the 15‑minute exposure period.

The quality criteria required for acceptance of results in the test were satisfied.

The test material was considered to be non-irritant according to the criteria of the Regulation (EC) No 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.