2-methylcyclohexanone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: 21 March 2019
Experimental Starting Date: 16 April 2019
Experimental Completion Date: 03 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

Final

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

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:

In the present study 2-MCH was dissolved in DMSO. Based on a molecular weight of 112.17 g/mol a stock solution of 200 mM was prepared.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Vehicle / solvent control:

DMSO

Negative control:

other: DMSO (AppliChem; Lot No.: 0001603375) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control.

Positive control:

cinnamic aldehyde [442D]

Results and discussion

In vitro / in chemico

Results

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.08

Cell viability:

Threshold validity of 70% cell viability/.
Cell viability Mean of 2 experiments - at least 92%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Acceptance Criteria
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Any other information on results incl. tables

Cytotoxicity
Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00 8.00 16.00 32.00 64.00	97.2 102.5 101.1 102.4 81.6	99.1 98.7 97.0 90.8 101.4	98.1 100.6 99.1 96.6 91.5	1.3 2.7 2.9 8.2 14.0
Test Item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	84.3 108.8 106.4 101.6 108.4 111.3 109.2 114.2 109.4 115.2 108.8 90.3	99.6 100.9 93.1 99.7 86.2 88.5 90.1 91.1 83.1 93.9 95.6 93.6	92.0 104.9 99.7 100.7 97.3 99.9 99.6 102.6 96.2 104.5 102.2 92.0	10.8 5.6 9.4 1.4 15.8 16.1 13.5 16.3 18.6 15.1 9.3 2.4

Luciferase Activity Experiment 1
Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Cell Viability [%]					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00 8.00 16.00 32.00 64.00	1.22 1.58 1.40 2.34 5.75	1.02 1.12 1.59 2.38 7.73	0.99 0.91 1.35 1.86 4.57	1.08 1.21 1.45 2.20 6.01	0.13 0.34 0.13 0.29 1.60	* *
Test Item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	0.91 0.85 1.04 0.92 0.99 0.85 0.93 1.02 0.96 1.06 1.02 1.15	0.71 0.87 0.82 0.75 0.80 0.87 0.81 0.77 0.58 0.81 0.78 0.70	0.70 0.80 0.84 0.72 0.87 0.82 0.74 0.82 0.88 0.77 0.82 0.68	0.78 0.84 0.90 0.80 0.88 0.85 0.83 0.87 0.81 0.88 0.87 0.84	0.12 0.04 0.12 0.11 0.10 0.02 0.10 0.13 0.20 0.15 0.13 0.27

* = significant induction according to Student’s t-test, p<0.05

See Figure 1: Evaluation of the KeratinoSens™ experiment 1 attached as a picture.

Luciferase Activity Experiment 2
Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Cell Viability [%]					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00 8.00 16.00 32.00 64.00	1.11 1.17 1.58 2.05 4.85	1.41 1.59 1.58 2.33 4.51	1.14 1.24 1.64 2.01 5.35	1.22 1.34 1.60 2.13 4.90	0.17 0.23 0.04 0.17 0.42	* * *
Test Item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	1.02 1.04 0.93 0.89 0.91 1.02 1.00 0.98 1.00 1.04 1.02 1.07	1.03 0.93 0.95 1.01 0.91 0.98 0.97 0.94 1.31 1.03 1.13 1.09	1.01 1.08 1.16 0.96 0.93 1.00 1.07 1.11 1.23 1.18 1.61 1.21	1.02 1.02 1.01 0.95 0.92 1.00 1.01 1.01 1.18 1.08 1.25 1.12	0.01 0.08 0.13 0.06 0.01 0.02 0.05 0.08 0.16 0.08 0.31 0.07

* = significant induction according to Student’s t-test, p<0.05

See Figure 2: Evaluation of the KeratinoSens™ experiment 2 attached as a picture.

Luciferase Activity - Overall Induction
Table 4: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00 8.00 16.00 32.00 64.00	1.08 1.21 1.45 2.20 6.01	1.22 1.34 1.60 2.13 4.90	1.15 1.27 1.52 2.16 5.46	0.10 0.09 0.11 0.05 0.79
Test Item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	0.78 0.84 0.90 0.80 0.88 0.85 0.83 0.87 0.81 0.88 0.87 0.84	1.02 1.02 1.01 0.95 0.92 1.00 1.01 1.01 1.18 1.08 1.25 1.12	0.90 0.93 0.96 0.88 0.90 0.92 0.92 0.94 1.00 0.98 1.06 0.98	0.17 0.12 0.08 0.11 0.02 0.11 0.13 0.10 0.26 0.14 0.27 0.20

See Figure 3: Overall Evaluation of the KeratinoSens™ Assay attached as a picture.

Additional Parameters
Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5 [µM]	n.a.	n.a.	n.a.	n.a.
Imax	0.90	1.25	1.08	0.25
IC30 [µM]	n.a.	n.a.	n.a.	n.a.
IC50 [µM]	n.a.	n.a.	n.a.	n.a.

Acceptance Criteria
Table 6: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	13.8	pass	17.3	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	3.0	pass
EC1.5 PC	± 2 x SD of historical mean	17.10	pass	12.94	pass
Induction PC at 64 µM	2 .00 < x < 8.00	6.01	pass	4.90	pass

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the
transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test
item can be considered as non-sensitiser.

Executive summary:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
In the present study 2-MCH was dissolved in DMSO. Based on a molecular weight of 112.17 g/mol a stock solution of 200 mM was prepared.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.
In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.
In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.
No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Under the condition of this study the test item is therefore considered as non-sensitiser.