2-methylcyclohexanone

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: 29 October 2019
Experimental Starting Date: 14 November 2019
Experimental Completion Date: 06 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Final

Deviations:

no

Remarks:

There were no deviations from the study plan.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-methylcyclohexanone

EC Number:

209-513-6

EC Name:

2-methylcyclohexanone

Cas Number:

583-60-8

Molecular formula:

C7H12O

IUPAC Name:

2-methylcyclohexan-1-one

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 2

Reference substance name:

Unknown impurities

Molecular formula:

not applicable

IUPAC Name:

Unknown impurities

Test material form:

liquid

Details on test material:

Batch No.: 01246M0002, purity confirmed by analytical certificate

Specific details on test material used for the study:

Prior to treatment, all EpiDerm™ tissues were gently blotted to remove moisture. The test item was applied undiluted. 30 µL (47 µL/cm2) of the test item were dispensed directly atop the EpiDerm™ tissue. The test item was gently spread to match size of the tissue using a bulb-headed Pasteur pipette.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test was carried out with the reconstituted three-dimensional human skin model EpiDerm™
(MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have
been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (MillicellR). The EpiDerm™ epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied undiluted. 30 µL (47 µL/cm2) of the test item were dispensed directly atop the EpiDerm tissue.

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

All plates were incubated for 25 ± 1 min under the sterile flow and for the remaining time of 35 ± 1 min transferred to the incubator. Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, in one-minute intervals. Subsequently, the inserts were completely submerged 3 times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip. The plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h. After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a 24-well plate containing 300 µL pre-warmed MTT medium.
This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
After the MTT incubation, the tissues were rinsed 3 times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for at least 2 h with gentle shaking on a plate shaker.
Before using the extracts, the plate had been shaken for 15 min and the inserts were pierced with an injection needle.The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate.OD was measured at 570 nm in a plate
spectrophotometer using isopropanol as a blank

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

4.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The test item showed irritant effects.

Any other information on results incl. tables

Pre-Experiments
The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the
solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
The mixture of 30 µL of the test item per 300 µl aqua dest. and/ per 300 µL isopropanol showed no
colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after
mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of
possible false-negative results were necessary.

Table 1. Result of the Test Item 2-MCH

Name	Negative Control					Positive Control					Test Item
Replicate Tissue	1		2		3	1		2	3		1		2	3
Absolute OD570	1.316		1.328		1.317	0.096		0.097	0.095		0.093		0.094	0.096
	1.312		1.315		1.315	0.094		0.097	0.100		0.097		0.101	0.103
Mean Absolute OD570	1.317****					0.097					0.097
OD570 – (Blank Corrected)	1.271		1.282		1.271	0.050		0.051	0.050		0.048		0.049	0.051
	1.266		1.270		1.270	0.049		0.051	0.055		0.051		0.055	0.057
Mean OD570 of the Duplicates (Blank Corrected)	1.268		1.276		1.270	0.049		0.051	0.052		0.050		0.052	0.054
Total Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	1.271*					0.051					0.052
SD of Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	0.004					0.002					0.002
Relative Tissue Viability [%]	99.8	100.3		99.9		3.9	4.0			4.1	3.9	4.1			4.2
Mean Relative Tissue Viability [%]	100.0					4.0**					4.1
SD of Relative Tissue Viability [%]***	0.3					0.1					0.2
CV [% Viabilities]	0.3					3.0					4.3

*
Blank-corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.
**
Mean relative tissue viability of the three positive control tissues is ≤ 20%.
***
Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.
****
The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects. The test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.

Executive summary:

In the present study the skin irritant potential of 2-MCH was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404, [7]) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.
The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
The mixture of 30 µL of the test item per 300 µl aqua dest. and/ per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.
The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (4.1%) after 60 min treatment and 42 h post-incubation.

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.316	1.328	1.317	0.096	0.097	0.095	0.093	0.094	0.096
	1.312	1.315	1.315	0.094	0.097	0.100	0.097	0.101	0.103
Mean Absolute OD570	1 317****			0.097			0.097
OD570 (Blank Corrected)	1.271	1.282	1.271	0.050	0.051	0.050	0.048	0.049	0.051
	1.266	1.270	1.270	0.049	0.051	0.055	0.051	0.055	0.057
Mean OD570 of the Duplicates (Blank Corrected)	1.268	1.276	1.270	0.049	0.051	0.052	0.050	0.052	0.054
Total Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	1.271*			0.051			0.052
SD of Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	0.004			0.002			0.002
Relative Tissue Viability [%]	99.8	100.3	99.9	3.9	4.0	4.1	3.9	4.1	4.2
Mean Relative Tissue Viability [%]	100.0			4.0**			4.1
SD of Relative Tissue Viability [%]***	0.3			0.1			0.2
CV [% Viabilities]	0.3			3.0			4.3