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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2, 2012 - April 29, 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylcyclohexyl acetate
EC Number:
227-231-1
EC Name:
2-methylcyclohexyl acetate
Cas Number:
5726-19-2
Molecular formula:
C9H16O2
IUPAC Name:
2-methylcyclohexyl acetate
Details on test material:
- Name of test material (as cited in study report): 2-methylcyclohexylacetate
- Physical state: liquid
- Analytical purity: 99.33% weight
- Lot/batch No.: DNTL/OMCHA/020212
- Expiration date of the lot/batch: 15 March 2013 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: At room temperature in the dark and dry (avoid moisture due to the reactivity)

In vitro test system

Test system:
human skin model
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no: 12-EKIN-016). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ºC
- Temperature of post-treatment incubation (if applicable): 37.0 ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all
tissues were dosed and rinsed.
- Observable damage in the tissue due to washing: none
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
44 h
Number of replicates:
3

Test system

Type of coverage:
open

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
>= 87.4 - <= 88.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100 ± 18
Positive controls validity:
valid
Remarks:
4 ± 4
Other effects / acceptance of results:
The relative mean tissue viability obtained after 15 minutes treatment with 2-methylcyclohexylacetate compared to the negative control tissues was 88%. Since the mean relative tissue viability for 2-methylcyclohexylacetate was above 50% 2-methylcyclohexyl acetate is considered to be non-irritant.

Any other information on results incl. tables

Mean tissue viability (percentage of control):

 

A

B

C

Mean ± SD

Negative control

79

111

110

100 ± 18

2-methylcyclohexyl acetate

88

93

82

88 ± 6

Positive control

5

3

4

4 ± 4

Mean absorption:

 

A

B

C

Mean ± SD

Negative control

0.614

0.864

0.861

0.780 ± 0.143

2-methylcyclohexyl acetate

0.686

0.728

0.638

0.684 ± 0.045

Positive control

0.040

0.026

0.032

0.033 ± 0.007

 

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

Acceptability of the assay:

The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control were within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability was <18.

The mean relative tissue viability of the positive control was <40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be was <18.

The SD calculated from individual % tissue viabilities of the three identically treated replicates was <18.

According to the technical data, safety sheet and certificate of analysis of the reconstructed human epidermis (data available from Skinethic), used skin met all acceptability criteria.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
2-methylcyclohexyl acetate was determined to be non-irritant in the in vitro skin irritation test on a human three dimensional epidermal model (EPISKIN Small Model).
Executive summary:

An in-vitro skin irritation test was performed on 2 -methylcyclohexyl acetate in accordance with OECD Guideline 439. 2-methylcyclohexylacetate was applied undiluted (25 μl) directly on top of the skin tissue (human three dimensional epidermal model (EPISKIN Small Model)) for 15 minutes. After a post-incubation period of approximately 44 hours, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with 2-methylcyclohexylacetate compared to the negative control tissues was 88%. Since the mean relative tissue viability was above 50% after 15 minutes treatment, 2-methylcyclohexylacetate was considered to be non-irritant.

According to the technical data, safety sheet and certificate of analysis of the reconstructed human epidermis (data available from Skinethic), used skin met all acceptability criteria.

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