4-methylcyclohexanone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2017 to 10 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC No 706/2017

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No chemical analysis of the dose formulation was performed. Formulations were prepared just prior to incubation. Therefore this is considered not to prejudice the overall GLP status of the study.

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The assay determines the chemical reactivity of the test item to cysteine and lysine peptides, which is a unifying characteristic of most skin sensitizing chemicals. Reactivity (% depletion) is determined following 24-hour contact between test item and peptide in acetonitrile at the ratios 1:10 cysteine:test item and 1:50 lysine:test item by liquid chromatography with Ultra-Violet detection.

Test material

Test material information

Constituent 1

Reference substance name:

4-methylcyclohexanone

EC Number:

209-665-3

EC Name:

4-methylcyclohexanone

Cas Number:

589-92-4

Molecular formula:

C7H12O

IUPAC Name:

4-methylcyclohexan-1-one

Test material form:

liquid

Details on test material:

- Other: Colourless liquid

In chemico test system

Details on the study design:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.

Based on solubility results, acetonitrile was used as the vehicle. The positive control was cinnamaldehyde.

Cysteine peptide (peptide sequence Ac-RFAACAA-COOH) and lysine peptide (peptide sequence AC-FRAAKAA-COOH) were used. The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution and the lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution.

Design of the Direct Peptide Reactivity Assay: The test item was tested in two runs (since borderline results were obtained in the first run). The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer:
- co-elution control with cysteine peptide (50 µL test item formulation incubated with 750 µL of cysteine peptide dilution buffer (without cysteine peptide) and 200 µL of acetonitrile);
- co-elution control with lysine peptide (250 µL of test item formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide));
- reference control C prepared with cysteine peptide (50 µL of vehicle (acetonitrile) was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile);
- reference control C prepared with lysine peptide (250 µL of vehicle (acetonitrile) was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2));
- cinnamaldehyde (positive control) with cysteine peptide (50 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile);
- cinnamaldehyde (positive control) with lysine peptide (250 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2));
- test item with cysteine peptide (50 µL of test item formulation was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile);
- test item with lysine peptide (250 µL of test item formulation was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2)).

All vials were capped and mixed by vortex. All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated up to 26 hours and 48 minutes for the first cysteine analytical sequence or 24 (± 2) hours at 25°C and protected from light before injection into the HPLC/UV system. At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation. Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

The study samples were assayed in batches using HPLC/UV analysis.

The percent peptide depletion was calculated. Each appropriate peak was integrated and the peak area for calibration standards, control and test item samples were determined. Based on the concentration of standards and their peak area, a linear calibration curve was generated. Then, the concentration of peptide was determined in each sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of peptide using the linear calibration curves. Then, for each positive control and test item replicate, the percent depletion of peptide was determined from the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent) by using the following formula:
% depletion = 1 - (peptide peak area in replicate injection/mean peptide peak area in relevant reference control C samples) x100
Then, the mean percent depletion of the three replicates was calculated for each peptide as well as the mean of the percent cysteine and percent lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean % depletion.

- Evaluation of the possible co-elution of the test item with the lysine or cysteine peptides: In order to detect possible co-elution of the test items with a peptide, chromatograms of the co-elution control samples were analysed and compared with those of the reference control C samples.

Results and discussion

Positive control results:

In both runs the positive control showed high reactivity (100 mM cinnamaldehyde first run, the mean depletion rate of cinnamaldehyde was 79.64% and in the second run was 76.58%)

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: Mean peptide depletion (%)

Remarks:

Mean of cysteine and lysine depletion rates

Value:

4.4

Positive controls validity:

valid

Remarks:

cinnamaldehyde

Remarks on result:

no indication of skin sensitisation

Remarks:

no/minimal reactivity

Results 2

Key result

Run / experiment:

other: 2

Parameter:

other: Mean peptide depletion (%)

Remarks:

Mean of cysteine and lysine depletion rates

Value:

5.17

Positive controls validity:

valid

Remarks:

cinnamaldehyde

Remarks on result:

no indication of skin sensitisation

Remarks:

no/minimal reactivity

Any other information on results incl. tables

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co‑elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described:

- for the cysteine peptide, the mean depletion value was 7.12% and 9.29% for the first and second runs, respectively

- for the lysine peptide, the mean depletion value was 1.68% and 1.06% for the first and second runs, respectively

- The mean of the percent cysteine and percent lysine depletions in the first run was equal to 4.40%. This value being < 6.38%, the test item was considered to have no or minimal reactivity, though with limitations due to test item precipitation with the cysteine peptide. However, since the mean percentage depletion fell within the range of 3% to 10% (i.e.4.40%),the negative results obtained with the test item were considered as borderline and a second run was performed in order to allow a firm conclusion.

 

The mean of the percent cysteine and percent lysine depletions in the second run was equal to 5.17%. This value being < 6.38%, the test item was considered to have no or minimal reactivity. Since the lack or minimal reactivity was reproduced in the second run, the overall results between the two runs performed allowed a firm negative conclusion, though with limitations due to test item precipitation with the peptides.

Table 1: Percent peptide depletion (first run)

Sample		Cysteine peptide (% depletion)	Lysine peptide (% depletion)	Mean depletion rate (%) test item	Depletion classification
100 mM test item	Mean % depletion	7.12 ± 2.96	1.68 ± 0.39	4.40	no/minimal reactivity
	% CV	41.5	23.5
	Precipitate	yes	no
	Micelle	no	no
100 mM cinnamaldehyde	Mean % depletion	97.16 ± 0.59	62.12 ± 0.99	79.64	high reactivity
	% CV	0.6	1.6

 

Table 2: Percent peptide depletion (second run)

Sample		Cysteine peptide (% depletion)	Lysine peptide (% depletion)	Mean depletion rate (%) test item	Depletion classification
100 mM test item	Mean % depletion	9.29 ± 1.78	1.06 ± 0.35	5.17	no/minimal reactivity
	% CV	19.2	33.1
	Precipitate	yes	yes
	Micelle	no	no
100 mM cinnamaldehyde	Mean % depletion	96.87 ± 0.14	56.30 ± 1.21	76.58	high reactivity
	% CV	0.1	2.1

 

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The DPRA test method cannot be used alone for the purposes of classification.

Conclusions:

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item was considered to have no/minimal peptide reactivity, and is likely not to have any potential to cause skin sensitization, though with limitations due to its precipitation with the cysteine peptide.

Executive summary:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion basedon the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

 

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co‑elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide.  

·     

The mean of the percent cysteine and percent lysine depletions in the first run was equal to 4.40%. This value being < 6.38%, the test item was considered to have no or minimal reactivity, though with limitations due to test item precipitation with the cysteine peptide. However, since the mean percentage depletion fell within the range of 3% to 10% (i.e.4.40%), the negative results obtained with the test item were considered as borderline and a second run was performed in order to allow a firm conclusion.

 

The mean of the percent cysteine and percent lysine depletions in the second run was equal to 5.17%. This value being < 6.38%, the test item was considered to have no or minimal reactivity. Since the lack or minimal reactivity was reproduced in the second run, the overall results between the two runs performed allowed a firm negative conclusion, though with limitations due to test item precipitation with the peptides.

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item was considered to have no/minimal peptide reactivity, and is likely not to have any potential to cause skin sensitization, though with limitations due to its precipitation with the cysteine peptide.