Reaction mass of 1-(3,3-dimethylcyclohexyl)ethanone and 2,6,6-trimethylcycloheptanone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November, 2016 - 16 December, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

1

Reference substance name:

1-(3,3-dimethylcyclohexyl)ethan-1-one

EC Number:

246-799-1

EC Name:

1-(3,3-dimethylcyclohexyl)ethan-1-one

Cas Number:

25304-14-7

Molecular formula:

C10H18O

IUPAC Name:

1-(3,3-dimethylcyclohexyl)ethanone

Test material form:

liquid

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Controls:
Positive control: ethylene dimethacrylate glycol, 31.25-500 µM, tested in triplicate
Negative control: DMSO (vehicle) (1% in exposure medium)
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values

Number of replicates: three independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

Test system:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages.
One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

Test item preparation:
No correction was made for the composition/purity of the test item.
The test item was dissolved in DMSO to a final concentration of 200 mM. The compound formed a clear colourless solution at 200 mM. From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series): 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977 mM.
The stock and spike solution were diluted 25-fold with exposure medium resulting in concentrations of 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.813 and
3.906 µM (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.976 µM. All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. Three independent experiments were performed.

Media:
Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 µg/ml).
Exposure medium
Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

Treatment of cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2.

Luciferase acitivity measurement:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).

Cytotoxicity assessment:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed (e.g. by adding 10% SDS solution to each well) overnight. After shaking, the absorption is measured with the TECAN Infinite® M200 Pro Plate Reader.

Data analysis:
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.

Equation 1: Fold induction= (Lsample - Lblank)/(Lsolvent - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control

The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.

Equation 2: EC1.5 = (Cb - Ca) x [(1.5 - Ia) / (Ib - Ia)] + Ca
Where:
Ca is the lowest concentration in μM with > 1.5 fold induction
Cb is the highest concentration in μM with < 1.5 fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

Viability is calculated by Equation 3:
Equation 3: Viability = 100 x (Vsample - Vblank) / (Vsolvent - V blank)
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

Data intepretation:
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative (Figure 1):
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
• The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 6 wells tested. If the variability is higher, results should be discarded.

Results and discussion

Positive control results:

Experiment 1: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.26 and the EC1.5 31.9 µM.
Experiment 2: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.69 and the EC1.5 27.0 µM.
Experiment 3: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.75 and the EC1.5 62.8 µM.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1, 2000 µM

Parameter:

other: Imax

Value:

2.22

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

no dose response was observed

Results 2

Key result

Run / experiment:

other: Experiment 2, 2000 µM

Parameter:

other: Imax

Value:

1.27

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: Experiment 3, 125 µM

Parameter:

other: Imax

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (12.7, 6.2% and 7.3% in experiment 1, 2 and 3 respectively).
- Acceptance criteria met for positive control: yes. The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
•The EC1.5 of the positive control was between 5 and 125 µM (31.9 µM, 27.0 µM and 62.8 µM in experiment 1, 2 and 3 respectively). A dose response was observed in all three experiments and the induction at 250 µM was >2-fold in experiment 1 and 3 and with 1.94-fold just below 2-fold in experiment 2.

- Acceptance criteria met for variability between replicate measurements: Experiment 1 was considered inconclusive. Luciferase induction >1.5-fold was observed in the first experiment at all test concentrations with exception at 1000 µM. The maximum luciferase activity induction (Imax) was 2.22-fold but no clear dose response was observed. No EC1.5 was calculated. The first experiment was concluded as inconclusive. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in experiment 2 and 3. The maximum induction was 1.27 and 1.10-fold in experiment 2 and 3, respectively. Therefore the test substance is classified as negative in Experiment 2 and 3.

Any other information on results incl. tables

Summary tables

Table 1. Overview luminescence induction and cell viability of the test substance in Experiments 1, 2 and 3

Concentration (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.50	125	250	500	1000	2000
Exp 1 luminescence	1.63***	1.62***	1.71***	1.69***	1.60***	1.60***	1.67***	1.68***	1.70***	1.74***	1.08	2.22***
Exp 1 viability (%)	101.6	121.4	118.2	119.7	103.1	106.1	108.4	111.7	109.0	113.1	176.5	100.5
Exp 2 luminescence	1.00	0.90	0.94	0.97	0.94	0.97	1.01	1.04	1.01	1.05	1.07	1.27
Exp 2 viability (%)	108.7	115.5	109.8	119.1	136.4	111.1	106.8	102.6	100.8	102.3	92.4	91.3
Exp 3 luminescence	0.92	0.99	1.00	1.03	1.03	1.05	1.03	1.10	1.06	0.94	0.98	1.09
Exp 3 viability (%)	102.0	107.9	93.7	109.3	103.1	66.5	84.8	74.8	78.9	91.5	100.1	87.8

*** p<0.001 Students t-test

Table 2. Overview of luminescence induction and cell viability positive controlEthylene dimethacrylate glycol in Experiments 1, 2 and 3

Concentration (µM)	7.81	15.6	31.3	62.5	125	250
Exp 1 luminescence	1.12	1.20	1.49	1.74***	2.20***	3.26***
Exp 1 viability (%)	86.5	89.2	94.8	96.0	99.7	105.2
Exp 2 luminescence	1.25	1.37	1.55**	1.75***	1.94***	2.69***
Exp 2 viability (%)	122.7	128.1	136.3	148.9	131.7	124.6
Exp 3 luminescence	0.97	1.02	1.23	1.50	1.86***	2.75***
Exp 3 viability (%)	107.3	92.7	92.4	107.1	106.4	107.6

** p<0.01; *** p<0.001 Students t-test

Table 3. Overview of EC1.5, IC30 and IC50 values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test substance Experiment 1	NA*	2.22	NA	NA
Test substance  Experiment 2	NA	1.27	NA	NA
Test substance Experiment 3	NA	1.10	NA	NA
Pos Control Experiment 1	31.9	3.26	NA	NA
Pos Control Experiment 2	27.0	2.69	NA	NA
Pos Control Experiment 3	62.8	2.75	NA	NA

N.A. = Not applicable

* No dose response but all test concentrations with exception of 1000 µM showed >1.5-fold induction

Applicant's summary and conclusion

Interpretation of results:

other: KeratinoSens assay was negative

Conclusions:

In a GLP-compliant guideline study, the test substance did not cause a biologically relevant induction in luciferase activity in Keratinosens assay. Based on this, the test substance is considered to give a negative result under the experimental conditions in this assay.

Executive summary:

The GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. Three independent experiments were performed. The test substance was tested in a concentration range of test concentrations of 0.98 – 2000 µM (2-fold dilution series). Ethylene diemthacrylate glycol was used as a positive control. The acceptance criteria were met. In the first experiment, statistically significant luciferase induction >1.5-fold was observed at all test concentrations with exception at 1000 µM. The maximum luciferase activity induction (I_max) was 2.22-fold but no clear dose response was observed. No EC_1.5 (concentration at which induction of luciferase activity is above 1.5 fold threshold) was calculated. The first experiment was concluded as inconclusive. No biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in experiment 2 and 3. The maximum induction was 1.27 and 1.10-fold in experiment 2 and 3, respectively. Therefore the test substance was considered negative in Experiment 2 and 3. Based on the KeratinoSens^TMprediction model (2 out of 3) it is concluded that the compound is negative in this assay.