2-methylcyclohexanone

Endpoint summary

Description of key information

The in chemico direct peptide reactivity assay (DPRA) and the in vitro KeratinoSens™ assay were performed in order to detect the sensitisation potential of the 2-MCH.

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
Based on a molecular weight of 112.17 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analyzed by HPLC.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separations were regarded as not relevant.
No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cacetonitrile).
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides.
The mean depletion of both peptides was ≤ 6.38% (2.48%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

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.
Based on a molecular weight of 112.17 g/mol a stock solution of 200 mM was prepared.
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 two experiments, no significant luciferase induction > 1.5 was found in the tested concentration ranges. 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.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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

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]

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.

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

 

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.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Study Initiation Date: 21 March 2019
Experimental Starting Date: 23 April 2019
Experimental Completion Date: 06 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

Final

Deviations:

yes

Remarks:

There was the following deviation from the study plan: • Concerning: Study director, study plan, p. 2, 6 Study Plan: Study Director: Dr. Claudia Steinert Report: Study Director: Dr. Helge Gehrke Reason: Project handover. This deviation did not influence t

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage.
The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Depletion of the Cysteine Peptide
Mean Peptide Depletion [%] of Positive Control: 69.25.
Depletion of the Lysine Peptide
Mean Peptide Depletion [%] of Positive Control: 62.70

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

4.95 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

Acceptance Criteria
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Pre-Experiments
Solubility of the test item was determined prior to the main experiment. The test item was soluble in acetonitrile. No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using acetonitrile. All test item solutions were freshly prepared immediately prior to use.

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separations were regarded as not relevant.

Results of the Cysteine Peptide Depletion

Table 4: Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	5.1200	0.1553	69.56	69.25	0.31	0.45
	5.1740	0.1569	69.24
	5.2240	0.1584	68.94
Test Item	16.0590	0.4834	4.52	4.95	1.05	21.12
	16.1160	0.4851	4.19
	15.7870	0.4752	6.14

Results of the Lysine Peptide Depletion
Table 5: Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	5.6490	0.1858	62.72	62.70	0.18	0.29
	5.6810	0.1869	62.51
	5.6270	0.1851	62.87
Test Item	15.3000	0.5026	0.00	0.00	0.00	n/a
	15.2620	0.5013	0.00
	15.2880	0.5022	0.00

Categorization of the Test Item
Based on the results of the peptide depletion, categorization according to the prediction model might be performed.
Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered.

Table 6: Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	2.48	Minimal Reactivity	negative	4.95	Minimal Reactivity	negative
Positive Control	65.97	High Reactivity	positive	69.25	Moderate Reactivity	positive

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both
peptides. The test item is considered as “non-sensitiser”. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
In the present study 2-MCH was dissolved in acetonitrile, based on the results of the pre-experiments. The test item was soluble in acetonitrile. No turbidity, precipitation and phase separation was observed for the test item solution.
Based on a molecular weight of 112.17 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separations were regarded as not relevant.
No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cacetonitrile).
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides.
The mean depletion of both peptides was ≤ 6.38% (2.48%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins) 2-methylcyclohexanone showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser” and does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.4.

According to OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method) 2-methylcyclohexanone showed no induction of 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 and does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.4.