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Diss Factsheets

Administrative data

Description of key information

The OECD has published an Adverse Outcome Pathway (AOP) for skin sensitization, which consists of four key events. Consequently, 4-methylcyclohexanone has been assessed for its skin sensitising potential in three in vitro studies addressing molecular interaction with skin proteins (OECD 442C), inflammatory response in keratinocytes (OECD 442D), and activation of dendritic cells (OECD 442E), in accordance with REACH, Annex VII, 8.3.1.


 


Under the experimental conditions of the direct peptide reactivity assay (Chevallier, 2018), the test item was considered to have no/minimal peptide reactivity, though with limitations due to its precipitation with the cysteine peptide. The study was conducted under GLP according to a guideline method, and consequently assigned a Klimisch reliability rating of one.


 


Under the experimental conditions of the KeratinoSens assay (Richez, 2018), the test item was negative and therefore was considered to have no potential to activate the Nrf2 transcription factor, and cause inflammatory response in keratinocytes. The study was conducted under GLP according to a guideline method, and consequently assigned a Klimisch reliability rating of one.


 


Under the experimental conditions of the h-CLAT study (Gerbeix, 2018), the test item was found to be positive for the activation of dendritic cells. The study was conducted under GLP according to a guideline method, and consequently assigned a Klimisch reliability rating of one.


 


Supporting in silico investigations indicated are no structural alerts for skin sensitisation in either the OECD Toolbox (4.2) or DEREK Nexus (6.0.1). There was a skin sensitisation prediction by CAESAR (1.1.4), however, inspection of related molecules in the training set suggested this was outside of the applicability domain. Most molecules also contained the aldehyde functional group, which is positively associated with skin sensitisation. As a result, the CAESAR prediction is considered to be of low reliability.


 


4-methylcyclohexanone was negative in the DPRA and KeratinoSens studies, but positive in the h-CLAT. Protein binding was not indicated in the OECD Toolbox analysis or the DPRA, which looks at the initial molecular initiating event of the AOP. The KeratinoSens assay produced a negative result indicating that 4-methylcyclohexanone does not cause an inflammatory response in keratinocytes, which addresses the second key event in the AOP. DEREK did not report any structural alerts for skin sensitization. There was a positive outcome in the h-CLAT study, however, this is the third key event in the AOP. There was a skin sensitisation alert in CAESAR, however, this was outside of the applicability domain and is considered to be of low reliability.


 


As a result, based on the assessment of the in vitro and supporting in silico analysis, and in accordance with REACH, Annex XI, 1.2, 4-methylcyclohexanone is considered unlikely to have skin sensitising potential. The available data is considered to be adequate, relevant and reliable for the purposes of risk assessment and classification. No further in vivo testing is considered to be required, in accordance with REACH, Annex VII, 8.3.2.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
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.
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.



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%)
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
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

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

 

 

 

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.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 2017 to 21 July 2017
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 Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC No 706/2017 (2017)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Not reported
Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design: This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence.

Potential skin sensitizers are applied to the cells at 12 different concentrations for a period of 48 hours. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. The luciferase reporter gene is under control of a single copy of the ARE-element of the human AKR1C2. The luciferase production is measured by flash luminescence.

In parallel, cytotoxicity is measured by a MTT reduction and is taken into consideration in the interpretation of the sensitisation results. This evaluation is performed in at least two independent runs.




Positive control results:
During both runs, the criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e. Imax values of 17.08 or 14.66 during the first and second runs respectively). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was always obtained for the positive control, this was considered not to have any impact on the validity of the results of these runs.
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Remarks:
average induction
Value:
1.45
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
No statistically significant gene-fold induction above the threshold of 1.5
Key result
Run / experiment:
other: 2
Parameter:
other: Imax
Remarks:
average induction
Value:
1.51
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
No statistically significant gene-fold induction above the threshold of 1.5
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

At concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO:

· No precipitate/emulsion was observed in any test item treated wells at the end of the 48-hour treatment period in either run.

· No noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70% in both runs).

· No statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations in either run. 

· For both runs, the Imax values were not statistically significant and were 1.45 and 1.51 in the first and second runs, respectively. Moreover, no EC1.5 was calculated.

No geometric mean IC30 or IC50 was calculated since the cell viability was >70% in both runs.

The evaluation criteria for a negative response were met in both runs, the final outcome is therefore negative.  This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.  It cannot be used on its own to conclude on a lack of skin sensitisation potential.

Interpretation of results:
study cannot be used for classification
Remarks:
The KeratinoSens assay cannot be used alone for the purposes of classification.
Conclusions:
Under the experimental conditions of this study, the test item was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.
Executive summary:

The objective of this study was to evaluate the potential of the test item, to activate the Nrf2 transcription factor.  This test is a part of a tiered strategy for the evaluation of skin sensitisation potential.  Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line.  The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter.  This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2.  The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C.  Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls.  The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence.  In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed. For each run, the test item was solubilised in DMSO at 200 mM.

All acceptance criteria were fulfilled for the positive and negative controls, in each run. Both runs were therefore considered to be valid. 

Both runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

· No precipitate/emulsion was observed in any test item treated wells at the end of the 48-hour treatment period in either run.

· No noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70% in both runs).

· No statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations in either run.

· For both runs, the Imax values were not statistically significant and were 1.45 and 1.51 in the first and second runs, respectively. Moreover, no EC1.5 was calculated.

No geometric mean IC30 or IC50 was calculated since the cell viability was > 70% in both runs.

The evaluation criteria for a negative response were met in both runs, the final outcome is therefore negative. This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a lack of skin sensitisation potential.

Under the experimental conditions of this study, the test item was negative in the KeratinoSens assay and, therefore, was considered to have no potential to activate the Nrf2 transcription factor.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 2017 to 21 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 442E
Version / remarks:
2016
Deviations:
yes
Remarks:
see "any other info on methods field"
Qualifier:
according to guideline
Guideline:
other: EC No. 2017/706
Version / remarks:
2017
Deviations:
yes
Remarks:
see "any other info on methods field"
Qualifier:
according to guideline
Guideline:
other: DB-ALM Protocol No. 158: human Cell Line Activation Test (h-CLAT)
Deviations:
yes
Remarks:
see "any other info on methods field"
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:
activation of dendritic cells
Justification for non-LLNA method:
The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic dendritic cell (DC) activation, which plays a critical role in T-cell priming. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The Relative Fluorescence Intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitizers and non-sensitizers.
Details on the study design:
- Vehicle and vehicle control: Dimethylsulphoxide (DMSO).
- Positive controls: 2,4-Dinitrochlorobenzene (DNCB), Nickel Sulfate (NiSO4)

Details on study design: Initially, a solubility assessment was first performed in 0.9% NaCl and DMSO to select the vehicle and highest concentration to be used for test item formulation preparations. Then a Dose-Range Finding assay (DRF) was performed to assess test item toxicity and, determine the CV75 i.e. the test item concentration that result in 75% cell viability compared to the vehicle control. Secondly, based on cytotoxicity data obtained from the DRF, a concentration series was tested in successive runs in the main tests.

-DRF consisted of 2 separate assays at the following final concentrations: 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 µg/mL. Test item stock solutions were prepared in DMSO and then diluted 250 fold into cRPMI to obtain working solutions. The working solutions were finally used for exposure by adding 500 µL of working solutions to the volume of THP-1 cell suspension in the plate (500 µL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate. The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37°C and 5% CO2.

At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were resuspended with 600 µL of FACS buffer. Finally, cells were resuspended in 200 µL FACS buffer and the plate was positioned into the plate-reader of the flow cytometer. A volume of 10 µL of Propidium Iodide (PI) solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µg/mL per well.

The PI uptake was analyzed using flow cytometry with the acquisition channel B3. A total of 10000 living cells (PI negative) were acquired. In case of low viability which does not allow obtaining 10000 living cells, a total of 30000 events is acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition.

- Main tests: There were 2 separate runs. Test item stock solutions were prepared at eight different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to 1.2-fold the mean CV75. The maximum concentration in the plates was 1000 µg/mL. All stock formulations were then 250-fold diluted into cRPMI to obtain working solutions. In parallel, the working solutions of positive controls DNCB and NiSO4 and vehicle control were prepared.

All working solutions were finally used for exposure by adding 500 µL of working solutions to the volume of THP-1 cell suspension in the plate (500 µL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate. The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37°C and 5% CO2.

During the main test, test item administration was performed at the following concentrations (final concentrations in the wells): 279.08, 334.90, 401.88, 482.25, 578.70, 694.44, 833.33, 1000 µg/mL.

At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL FACS buffer and blocked with 600 µL of blocking solution and incubated at 4 °C for 15 minutes (± 1 minute). After blocking, cells were split in three aliquots of 180 µL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 minutes (± 2 minutes) at 4°C. Finally, cells were washed with 150 µL FACS buffer two times and resuspended in 200 µL FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 µL of PI solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µg/mL per well.

The expression of IgG1, CD86 and CD54 was analyzed by flow cytometry with the acquisition channel B1 in order to obtain the Mean Fluorescence Intensity (MFI); whereas the viability (PI uptake) was analyzed with the acquisition channel B3. A total of 10000 living cells (PI negative) were acquired. When the viability was low and did not allow obtaining 10000 living cells, a total of 30000 events was acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition. In case cell viability is less than 50%, no MFI is presented in the study report and the corresponding test item concentration are considered too high for interpretation because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction. Based on the Mean Fluorescence Intensity (MFI), the Relative Fluorescence Intensity (RFI) of CD86 and CD54 were calculated.

A run conclusion is positive if at least one of the conditions below is met:
• RFI of CD86 is ≥ 150 at any concentration leading to ≥ 50% viability,
• RFI of CD54 is ≥ 200 at any concentration leading to ≥ 50% viability.
In other circumstances, the run is considered as negative.

• If the first two runs are both positive for CD86 and/or are both positive for CD54, the h CLAT prediction is considered POSITIVE and a third run does not need to be conducted,
• If the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE (with due consideration of the highest-tested dose conditions) without the need for a third run,
• If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE.
• Results from the present study can be used to support the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in the context of Integrated Approaches to Testing and Assessment (IATA). However, results obtained at completion of the study will not be usable on their own, neither to sub-categorize skin sensitizers into sub categories 1A and 1B as defined by UN GHS, for authorities implementing these two optional sub-categories, nor to predict potency for safety assessment decisions.


Positive control results:
100 µg/mL NiSO4 positive in CD86 and CD54
4.0 µg/mL DNCB positive in CD86 and CD54
Key result
Run / experiment:
other: A
Parameter:
other: General conclusion
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
positive in CD54
Key result
Run / experiment:
other: B
Parameter:
other: General conclusion
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
positive in CD86 and CD54

DRF results:

- DRF first test: At post-treatment observation, no abnormalities such as precipitate/emulsion or cell morphology modification was observed at any tested concentrations; flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75% at the concentration of 1000 μg/mL. The corresponding CV75 value was 914.43 μg/mL.

- DRF second test: At post-treatment observation, no abnormalities such as precipitate/emulsion or cell morphology modification was observed at any tested concentrations; flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75% at the concentration of 1000 μg/mL. The corresponding CV75 value was 813.62 μg/mL.

- Based on the results from both DRF runs, the mean CV75 was 864.02 μg/mL. The calculated highest concentration (i.e. 1.2-fold the mean CV75) for use in the main test was, therefore, greater than the highest concentration tested in the DRF phase (i.e. 1000 μg/mL).

According to the OECD guideline, the highest tested concentration to be tested in the main test was set to 1000 μg/mL

Main test Run A:

- post-treatment observations: no precipitate/emulsion was noted in treated wells,

- no cytotoxic effect (i.e. cell viability < 50%) was noted at any tested concentration, but slight toxicity was noted at concentrations ≥694.44 μg/mL with viabilities ranging from 59.9 and 76.3% ,

- RFI CD86 did not exceed the positivity threshold at any concentrations,

- RFI CD54 exceeded the positivity threshold at the concentration of 401.88 μg/mL,

- the run was therefore considered positive for CD54.

Main test Run B:

- post-treatment observations: no precipitate/emulsion was noted in treated wells,

- no cytotoxic effect (i.e. cell viability < 50%) was noted at any tested concentration, but slight toxicity was noted at the concentrations ≥833.33 μg/mL with viabilities ranging from 57.3 and 78.6%,

- RFI CD86 exceeded the positivity threshold at the concentrations of 401.88, 833.33 and 1000 μg/mL,

- RFI CD54 exceeded the positivity threshold at concentrations ≥ 482.25 μg/mL,

- the run was therefore considered positive for CD 86 and CD54.

Table 1: Summary results of main test results

Conc (μg/mL)

RFI for CD86

RFI for CD86

RFI for CD54

RFI for CD54

Viability (%)

Viability (%)

 

A

B

A

B

A

B

279.08

107

125

185

162

91.1

94.6

334.90

132

141

193

108

90.7

93.8

401.88

132

194

211

169

88.1

91.5

482.25

118

139

170

246

88.8

92.7

578.70

111

130

130

262

89.0

93.1

694.44

138

130

193

292

73.6

86.0

833.33

104

158

163

377

66.8

78.6

1000.00

127

161

130

331

59.9

57.3

Run A conclusion: run with positive outcome in CD54

Run B conclusion: run with positive outcome both CD86 and CD54

Interpretation of results:
study cannot be used for classification
Remarks:
The h-CLAT test method cannot be used alone for the purposes of classification.
Conclusions:
Under the experimental conditions of this study, the test item was found to be positive in the h-CLAT.
Executive summary:

The objective of the study was to determine the ability of the test item to induce an increase in cell surface markers expression in THP-1 cells using the h-CLAT test method.

Following a solubility assessment and a Dose-Range Finding assay (to select sub-toxic concentrations for testing in the main test), the skin sensitizing potential of the test item was tested in the main test in two successive runs. In each run, the test item formulations were applied to triplicate wells containing THP-1 cells and cultured for 24 hours ± 30 minutes at 37°C, 5% CO2 in a humidified incubator. A set of control wells (each in triplicate) was also added in each plate to guarantee the validity of each run. At the end of the incubation period, all cells from the first wells were labelled with IgG1-FITC antibodies, the second ones were labelled with CD86-FITC antibodies and the third ones were labelled with CD54-FITC antibodies. Then, just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination. For each run, the Mean Fluorescence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. The corrected MFI value from the corresponding vehicle control was set to 100% CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

The test item was soluble in DMSO at the concentration of 500 mg/mL. The test item induced a decrease in cell viability <75% in both DRF runs and the calculated mean CV75 value was: 864.02 μg/mL. The calculated highest concentration (i.e. 1.2-fold the mean CV75) for use in the main test was therefore greater than the highest concentration tested in the DRF phase (i.e. 1000 μg/mL). According to the OECD guideline, the highest tested concentration to be tested in the main test was set to 1000 μg/mL.

In Run A, there was a positive outcome in CD54 and in Run B, there was a positive outcome in both CD86 and CD54.

Under the experimental conditions of this study, the test item was found to be positive in the h-CLAT.

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

Based on the weight of evidence assessment from three in vitro studies addressing separate key steps in the AOP, and supporting in silico analysis, 4-methylcyclohexanone is considered to be not classified for skin sensitisation.