2-Propenoic acid, 2-[[1,1,2-trifluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)ethyl]thio]ethyl ester

Endpoint summary

Description of key information

Based on the results of an in chemico test (DPRA, OECD TG 442C) the test item is peptide reactive. The test item is considered positive for the first key event of the skin sensitisation Adverse Outcome Pathway (AOP) (reference 7.4.1-1).

The test item induced the luciferase activity in the transgenic KeratinoSens™ cell line under the test conditions of the study. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP) (reference 7.4.1-2).

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:

2019-10-14 to 2019-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method: B.60 (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

14 February 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution
The test item was dissolved in dimethyl sulfoxide (DMSO). A stock solution of 200 mM was prepared by pre-weighing the test item into 4 mL glass vessels.

- Preparation of the test chemical serial dilutions
Based on the stock Solution a set of twelve master Solutions in 100% DMSO were prepared. The stock solution (200 mM) of the test items were diluted eleven times using a constant dilution factor of 1:2 (100 µL stock Solution + 100 µL DMSO). Then the 100x concentrated master solutions were further diluted 1:25 in test item exposure medium (10 µL + 240 µL test item exposure medium) resulting in a 4% share of the solvent. These 4-fold concentrated test item solutions were finally diluted 1:4 when incubated with the cells (50 µL + 150 µL test item exposure medium). Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

- Preparation of the positive controls
Cinnamic aldehyde was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM - 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test items, resulting in a final concentration range of 4 µM - 64 µM. The final concentration of DMSO was 1% (v/v) for all wells.

- Preparation of the solvent and blank controls
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells filled with the negative control were included in every testing plate. The preparation of the negative control was carried out analogous to the test item. A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3 (test item and positive control); 6 (solvent control)
- Number of repetitions: 2
- Test chemical concentrations: 0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM
- Application procedure
- Exposure time: 48 hours
- Description on study acceptance criteria
The test met 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 (7 µM - 30 µM based on the Validation dataset)
- the average coefficient of Variation (CV) of the luminescence reading for the negative (solvent) control is <20% in each repetition which is consisting of 6 wells.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density)
A cell suspension of 8 x 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 x 10^4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (Hat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.

- Incubation conditions: 37°C ± 1 °C and 5% CO2

- Washing conditions: After seeding, cells were grown for 24 ± 1h in assay medium at 37 ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.

- Precipitation noted: No

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer: Multi-Mode Microplate Reader (Cytation 1, BioTek Instruments GmbH, Bad Friedrichshall, Germany).
- Plate used: 96 well plate
- Lysate preparation: After washing 20 µL of passive lysis buffer was added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate was injected by the injector of the plate reader. The plate reader waited for 1 s before assessing the luciferase activity for 2 s. This procedure was repeated for each individual well.

DATA EVALUATION
- Cytotoxicity assessment
2.7 mL of a MTT Solution (5 mg/mL in DPBS) was added to 20 mL test item exposure medium. For the cell viability assay plate, the medium was replaced with 200 µL of this fresh medium containing MTT. The plate was covered with a sealing tape and incubated for 4h at 37 ± 1°C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 ± 1°C and 5% CO2 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at 600 nm.

- Prediction model used
A KeratinoSens™ prediction was considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is > 1.5-fold increased and statistically significant (p<0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity > 1.5-fold (i.e. at the EC1.5 determining concentration)
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction


If in a given repetition, all of the three first conditions are met but a clear dose-dependent increase in luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, a negative result obtained with test chemicals tested at a maximal test concentration <1000 µM (or 200 µg/mL for test chemicals with no defined MW) and which do not reach cytotoxicity (<70% viability) at the maximal tested concentration should also be considered as inconclusive. A negative result for test items with a log Kow >7 should be interpreted with care due to the applicability of the test method.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The average luciferase activity induction obtained with the positive control cinnamic aldehyde at the highest tested concentration 64 μM was 2.83 at Run 1 and 2.49 at Run 2.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

7.81 µM

Cell viability:

108.78 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

7.82

Cell viability:

108.78 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

9.91 µM

Cell viability:

134.27 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

6.59

Cell viability:

134.27 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes, the average coefficient of Variation (CV) of the luminescence reading is <20% in each repetition which is consisting of 6 wells (Run 1: 19.2 %; Run 2: 13.1 %)
- Acceptance criteria met for positive control: yes, the luciferase activity induction is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations and the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8 (Run 1: 2.83; Run 2: 1.52).
The EC1.5 value of the positive control is within two standard deviations of the historical mean; 7 µM - 30 µM based on the Validation dataset. (Run 1: 30 µM; Run 2: 16 µM).

Interpretation of results:

other: positive for the second key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

The test item induced the luciferase activity in the transgenic KeratinoSens™ cell line under the test conditions of the study. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed according to OECD 442D. It is used to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization Adverse Outcome Pathway) of the test item. In the present study the test substance was dissolved in DMSO. Based on a molecular weight of 398.22 g/mol a stock Solution of 200 mM was prepared. The cells were treated with different concentrations (0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM) of the test item and the luciferase gene induction was measured using light producing luciferase substrate. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from cells.

Test chemicals are considered positive in the KeratinoSens™ test method if they induce a statistically significant induction of the luciferase activity above a given threshold (>1.5 fold), below a defined concentration which does not significantly affect cell viability (< 1000 uM) and at a concentration at which the cellular viability is above 70%. For this purpose, the maximal fold induction of the luciferase activity over solvent control (Imax) was determined. Furthermore, since cells were exposed to series of concentrations of the test chemical, the concentration needed for a statistically significant induction of luciferase activity above the threshold (EC1.5 value) was interpolated from the dose-response curve obtained from the series of tested concentrations of the test chemical. Finally, parallel cytotoxicity measurements were conducted to assess whether luciferase induction occurs at sub-cytotoxic concentrations.

In the first run, a max luciferase activity induction (Imax) of 7.82 was determined. The lowest tested concentration with a luciferase induction >1.5 (2.10) was found to be 15.63 µM, the corresponding cell viability was >70% (108.78%). The calculated EC1.5 value was 7.81 µM. In the second run, a max luciferase activity induction (Imax) of 6.59 was determined. The lowest tested concentration with a luciferase induction >1.5 (1.80) was found to be 15.63 µM, the corresponding cell viability was >70% (134.27%). The calculated EC1.5 value was 9.91 µM. A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-12-10 to 2019-12-17

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:

June 18, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions
21.58 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (41.84 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
30.10 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (55.74 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

- Preparation of the test chemical solutions
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.

- Preparation of the positive controls, reference controls and co-elution controls

Positive controls: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

Co-elution controls: were set up in parallel to sample preparation but without the respective peptide solution. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

Reference control:
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A: was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run.
Reference control B: was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.
Reference control C: was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

INCUBATION
- Incubation conditions: The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel.
- Precipitation noted:
For the 100 mM stock solution of the test item phase separation was observed when diluted with the cysteine peptide solution. After the 24 ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation and precipitation (on the bottom of the HPLC vials) was observed for the samples of the test item (for the co-elution control only phase separation was observed). Samples of the test item and peptide were not centrifuged prior to the HPLC analysis, but pipetted into another vial.
For the 100 mM stock solution of the test item turbidity and phase separation was observed when diluted with the lysine peptide solution. After the 24 ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Slight 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 separation was regarded as not relevant.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys:
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions covering the range of 0 – 0.534 mM.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm. The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control.


Evaluation criteria of results according to Prediction model 1 (cysteine 1:10 / lysine 1:50)
Mean peptide depletion [%] Reactivity Evaluation
> 42.47; high reactivity; positive
> 22.62 ≤ 42.47; moderate reactivity; positive
> 6.38 ≤ 22.62; low reactivity; positive
0 ≤ 6.385; minimal or no reactivity; negative

Evaluation criteria of results according to prediction model 2 (cysteine 1:10).
mean Cys peptide depletion [%]; Reactivity; Evaluation
> 98.24 - ≤ 100; high reactivity; positive
> 23.09 ≤ 98.24; moderate reactivity; positive
> 13.89 ≤ 23.09; low reactivity; positive
0 ≤ 13.89; minimal or no reactivity; negative

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 70.36%.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

19.06 %

At concentration:

0.408 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

96.81 %

At concentration:

0.018 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

high reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes
- Acceptance criteria met for variability between replicate measurements: yes

Depletion of the Cysteine Peptide

Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.4600	0.1468	71.81	72.16	0.34	0.47
	4.4030	0.1450	72.18
	4.3520	0.1433	72.50
Test Item	0.4960	0.0174	96.87	96.81	0.06	0.06
	0.5040	0.0177	96.81
	0.5150	0.0180	96.75

 

Depletion of the Lysine Peptide

Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.2230	0.1601	68.23	68.55	0.28	0.41
	4.1650	0.1579	68.67
	4.1520	0.1574	68.76
Test Item	11.0020	0.4169	17.23	19.06	1.78	9.33
	10.7440	0.4071	19.17
	10.5300	0.3990	20.78

 

Interpretation of results:

other: positive for the first key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

The DPRA prediction is “positive” with high reactivity according to the prediction model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50). It can be considered positive for the first key event of the skin sensitisation Adverse Outcome Pathway.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) according to OECD TG 442C 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 the test item was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 398.22 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.

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item phase separation was observed when diluted with the cysteine peptide solution. After the 24 ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation and precipitation was observed for the samples of the test item (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis, but pipetted into another vial.

For the 100 mM stock solution of the test item turbidity and phase separation was observed when diluted with the lysine peptide solution. After the 24 ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Slight 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 separation was regarded as not relevant.

Phase separation and precipitates of the test item with both peptide peaks and additionally turbidity with the lysine peptide peak was observed. Therefore, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (57.93%). Even though precipitates and phase separation was observed a positive result can still be used. Based on the prediction model 1 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 70.36%.

In this study under the given conditions the test item showed high reactivity towards both peptides. The test item is considered positive for the first key event of the skin sensitisation Adverse Outcome Pathway.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

DPRA (reference 7.4.1-1)

The in chemico direct peptide reactivity assay (DPRA) according to OECD TG 442C 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 the test item was dissolved in acetonitrile, based on the results of the pre-experiments. 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.

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item, phase separation was observed when diluted with the cysteine peptide solution. After the 24 ± 2 h incubation period but prior to the HPLC analysis, samples were inspected for precipitation, turbidity or phase separation. Phase separation and precipitation was observed for the samples of the test item (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis, but pipetted into another vial.

For the 100 mM stock solution of the test item turbidity and phase separation was observed when diluted with the lysine peptide solution. After the 24 ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Slight 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 separation was regarded as not relevant.

Phase separation and precipitates of the test item with both peptide peaks and additionally turbidity with the lysine peptide peak was observed. Therefore, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (57.93%). Even though precipitates and phase separation was observed a positive result can still be concluded. Based on the prediction model 1 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 70.36%.

In this study under the given conditions the test item showed high reactivity towards both peptides. The test item is considered as “sensitiser”.

 

KeratinoSens (reference 7.4.1-2)

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed according to OECD 442D. It is used to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization Adverse Outcome Pathway) of the test item. In the present study the test substance was dissolved in DMSO. Based on a molecular weight of 398.22 g/mol a stock solution of 200 mM was prepared. The cells were treated with different concentrations (0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM) of the test item and the luciferase gene induction was measured using light producing luciferase substrate. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from cells.

Test chemicals are considered positive in the KeratinoSens™ test method if they induce a statistically significant induction of the luciferase activity above a given threshold (>1.5 fold), below a defined concentration which does not significantly affect cell viability (< 1000 uM) and at a concentration at which the cellular viability is above 70%. For this purpose, the maximal fold induction of the luciferase activity over solvent control (Imax) was determined. Furthermore, since cells were exposed to series of concentrations of the test chemical, the concentration needed for a statistically significant induction of luciferase activity above the threshold (EC1.5 value) was interpolated from the dose-response curve obtained from the series of tested concentrations of the test chemical. Finally, parallel cytotoxicity measurements were conducted to assess whether luciferase induction occurs at sub-cytotoxic concentrations.

In the first run, a max luciferase activity induction (Imax) of 7.82 was determined. The lowest tested concentration with a luciferase induction >1.5 (2.10) was found to be 15.63 µM, the corresponding cell viability was >70% (108.78%). The calculated EC1.5 value was 7.81 µM. In the second run, a max luciferase activity induction (Imax) of 6.59 was determined. The lowest tested concentration with a luciferase induction >1.5 (1.80) was found to be 15.63 µM, the corresponding cell viability was >70% (134.27%). The calculated EC1.5 value was 9.91 µM. A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

 

Conclusion

Based on the results of an in chemico/in vitro test strategy the test item shows covalent binding to proteins (DPRA, OECD 442C) and it induces the luciferase activity in keratinocytes (KeratinoSens™, OECD TG 442D) As both the first and second key event of the skin sensitisation Adverse Outcome Pathway (AOP) were considered positive, the substance is predicted to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is classified as skin sensitising Cat 1 (H317) according to Regulation (EC) No 1272/2008 (CLP).