4,4'-bis(chloromethyl)-1,1'-biphenyl

Endpoint summary

Description of key information

Chavallier (2017)

Under the experimental conditions of this study, the test item was considered to have a low peptide reactivity, though with limitations due to test item precipitation with the peptides. The test item is considered positive in the DPRA assay.

Michel (2017)

Under the experimental conditions of this study, the test material was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 January 2017 to 31 January 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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (acetone) at 100 mM. This formulation had the aspect of a clear colorless solution. The formulation was used just after its preparation.

Details on the study design:

VEHICLE
Several vehicles were tested during the solubility assay. The test item was found not soluble at 100 mM in acetonitrile, milli-Q water, 1:1 mixture of acetonitrile:milli-Q water and isopropanol, even after a sonication step of 1 minute. A solution was obtained at 100 mM in acetone (without sonication step). Therefore, this vehicle (acetone) was retained.
Based on solubility results, the retained vehicle was acetone.

POSITIVE CONTROL
The positive control was cinnamaldehyde. The positive control was dissolved in acetonitrile at 100 mM. The physical aspect of the formulation was clear colorless solution. The formulation was used just after its preparation.

CO-ELUTION CONTROL SAMPLES
In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.

REFERENCE CONTROL SAMPLES
For each peptide, the analytical batch included reference control samples (sub-categorized in reference control A, B or C samples). All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent. These samples were used to:
. Reference control A: Check the accuracy of the calibration curve for peptide quantification,
. Reference control B: Check the stability of the peptide during analysis,
. Reference control C: Check that the solvent did not impact the percentage of peptide depletion.

CYSTEINE PEPTIDE
. Peptide sequence: Ac-RFAACAA-COOH
. Peptide sequence synonyms: Ac RFAACAA-OH or RFAACAA-COOH
. Molecular weight: 750.88 g/mol
. Supplier: JPT Peptide Technologies GmbH
. Batch No.: 260515HSDWW0715
. Storage condition: At -20 °C
. Description: White powder

The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution.

LYSINE PEPTIDE
. Peptide sequence: Ac-RFAAKAA-COOH
. Peptide sequence synonyms: Ac RFAAKAA-OH or RFAAKAA-COOH
. Molecular weight: 775.91 g/mol
. Supplier: JPT Peptide Technologies GmbH
. Batch No.: 220114HSDWW0715
. Storage condition: At -20 °C
. Description: White powder

The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution.

EQUIPMENT, REAGENTS AND COMPUTER SYSTEMS
. High Performance Liquid Chromatography (HPLC) Systems with a UV detector (220 nm),
. Analytical chromatographic columns (Zorbax SB C18, 100 x 2.1 mm; 3.5 μm HPLC analytical column),


DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test item was tested in one run. The run was processed as follows.
- Preparation of the samples
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 samples preparation
For the co-elution control with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.

- For the co-elution control with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

- Reference control samples preparation
Reference control A and B samples: In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.

Reference control C samples: Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.

For the reference control C prepared with cysteine peptide: 50 μL of each vehicle (acetone or 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.

For the reference control C prepared with lysine peptide: In parallel, 250 μL of each vehicle (acetone or 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) depletion control samples preparation
For the reactivity of cinnamaldehyde 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.

For the reactivity of cinnamaldehyde with lysine peptide: In parallel, 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 samples preparation
For the reactivity of 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.

For the reactivity of test item with lysine peptide: In parallel, 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).

- Incubation of the samples
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 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 400 g 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.

- Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20 % acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.
The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

- HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least:
. one blank sample (peptide dilution buffer),
. one calibration curve injected at the beginning of the analytical batch,
. three reference control A samples,
. the co-elution control sample,
. three reference control B samples,
. reference control C sample (replicate 1),
. positive control sample (replicate 1),
. test item study sample (replicate 1),

The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3: Three reference control B samples.

HPLC CONDITIONS
The HPLC/UV method used for the samples analysis is summarized in the table below:
Analytical Column: Zorbax SB C18, 100 x 2.1 mm, 3.5 μm (Waters)
In-line filter C18, 4.0 x 2.0 mm (Phenomenex)
Mobile phase:
Mobile phase A: acetonitrile + 0.085 % TFA
Mobile phase B: milli-Q water + 0.1 % TFA
- Flow: 350 μL/minute
- Gradient:
Time % Mobile phase A % Mobile phase B
0 10 90
10 25 75
11 90 10
13 90 10
13.5 10 90
20 10 90

UV Wavelenght: 220 nm
Rinse solution: Acetonitrile
Oven temperature: 30.0 °C
Autosampler temperature: Nominal temperature of +25 °C
Injection volume: 5 μL
Retention times:
Cysteine-peptide: Approx. 9.8 minutes
Lysine-peptide: Approx. 7.5 minutes
Total analysis time: 20 minutes

DATA ANALYSIS AND CALCULATION
- Calculation of the percent peptide depletion
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)) x 100

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.
Peak areas and peptide concentrations are presented in the report. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated and reported.

- 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 analyzed and compared with those of the reference control C samples.

Key result

Remarks on result:

positive indication of skin sensitisation

Evaluation of the Presence of Precipitate at the end of the Incubation with Peptides

At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis.

As precipitate was observed in the test item, positive control and reference control samples incubated with the cysteine and lysine peptides as well as in co-elution samples prepared with the lysine or cysteine dilution buffer, these vials were centrifuged at 400 g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Thus, only supernatants were injected into the HPLC/UV system.

Evaluation of the Results

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 (% interference < 10 %). As a result, the mean percent depletion values were calculated for each peptide:

. for the cysteine peptide, the mean depletion value was 29.59 %,

. for the lysine peptide, the mean depletion value was 0.00 %.

The mean of the percent cysteine and percent lysine depletions was equal to 14.80 %. However, since precipitates were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated.

Since the mean of the percent cysteine and percent lysine depletions was between 6.38 and 22.62 %, the test item was considered to have a low peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization, though with limitations due to test item precipitation with the peptides.

Table 1. Determination of Cysteine Peptide and Lysine Peptide Depletion in Samples Spiked with a Solution at 100 mM of Test Material.

Sample number	Cysteine peptide		Lysine peptide		Mean depletion rate (%) of test material	Depletion classification
	Peak area (μV/sec)	% depletion	Peak area (μV/sec)	% depletion
1 2 3	1228848 1201290 1158729	27.68 29.30 31.80	1551039 1553036 1552155	0.00* 0.00* 0.00*
Mean SD % CV	- - -	29.59 2.08 7.0	- - -	0.00* nc nc	14.80	Low reactivity
Precipitate:	Yes		Yes
Micelle	No		No

*: Value set to 0 due to negative depletion

-: not applicable

Table 2. Acetonitrile Determination of Cysteine Peptide and Lysine Peptide Depletion in Samples Spiked With a Solution at 100 mM of Cinnamaldehyde.

Sample number	Cysteine peptide		Lysine peptide		Mean depletion rate (%) of Cinnamaldehyde	Depletion classification
	Peak area (μV/sec)	% depletion	Peak area (μV/sec)	% depletion
1 2 3	493840 514914 513144	72.95 71.79 71.89	625969 602903 65378	57.51 59.07 58.23
Mean SD % CV	- - -	72.21 0.64 0.9	- - -	58.27 0.78 1.3	65.24	High reactivity

Interpretation of results:

other: Classification will be determined in conjuction with other sensitisation studies as part of an integrated testing strategy.

Conclusions:

Under the conditions of this study, the test item was considered to have a low peptide reactivity and may therefore have the potential to cause skin sensitisation, though with limitations due to test item precipitation with the peptides.

Executive summary:

The potential of the test material to act as a sensitiser was investigated in a GLP study in accordance with the standardised guidelines OECD 442C.

This test was conducted as part of a tiered strategy for skin sensitization assessment. 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 based on 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 test item was dissolved at 100 mM in acetone.

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 (% interference < 10 %). As a result, the mean percent depletion values were calculated for each peptide using the formula described in Data analysis and calculation. For the cysteine peptide, the mean depletion value was 29.59 % and for the lysine peptide, the mean depletion value was 0.00 %.

The mean of the percent cysteine and percent lysine depletions was equal to 14.80 %. However, since precipitates were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated.

Since the mean of the percent cysteine and percent lysine depletions was between 6.38 and 22.62 %, the test item was considered to have a low peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization, though with limitations due to test item precipitation with the peptides.

Under the experimental conditions of this study, the test item was considered to have a low peptide reactivity, though with limitations due to test item precipitation with the peptides. The test item is considered positive in the DPRA assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2017 to 10 February 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

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
On the basis of solubility results, the test material was dissolved in DMSO at 200 mM. One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of two for the first run and a dilution factor of 1.41 for the second run (due to the high cytotoxicity observed in the first run) to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level. All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

Details on the study design:

TEST SYSTEM
KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test material of the Nrf2 transcription factor in this test.
. Supplier: Givaudan.
. Batch: C1.
. Storage condition: In the freezer set at -80 °C.
. Mycoplasma: absence of mycoplasma was confirmed.

SPECIFIC EQUIPMENT AND MEDIA
- Specific equipment: 96-well plate Luminometer with injectors and optical density reader (Varioskan Flash).
- Media:
Maintenance medium No. 1: DMEM containing GlutaMAXTM, 1000 mg/L D-Glucose, Sodium Pyruvate and supplemented with 9.1 % Fetal Calf Serum (FCS) and 500 μg/mL G-418.
Maintenance medium No. 2: DMEM with 9.1 % FCS without G-418.
Treatment medium: DMEM with 1 % FCS without G-418.
Freezing medium: DMEM with 20 % FCS and 10 % DMSO.

STUDY DESIGN
The test material was tested in two independent runs using cells from a different passage number.
- Solubility assay: A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water or treatment culture medium). Since the test material was found soluble in DMSO at 200 mM, this stock formulation was diluted in treatment culture medium to the final concentration of 2000 μM. Then, a visual inspection of the sample was performed to evaluate the presence of precipitate.

KERATINOSENS ASSAY
-Cell seeding for testing
Cells were grown using general culture procedures up to 80-90 % confluence. The day prior to treatment, cells were washed twice with D-PBS containing 0.05 % EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 10^4 cells/mL, cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 10^4 cells) per well taking care to avoid sedimentation of the cells during seeding. After seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test material addition.

-Treatment
After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium. From the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation. All plates were covered by a sealing membrane to avoid evaporation of volatile test materials and to avoid cross-contamination between wells. The plates were then incubated for 48 ± 2 hours at 37 °C, 5 % CO2, 90 % humidity.

ENDPOINT MEASUREMENTS
-Microscopic observation to evaluate the presence or absence of precipitate - transparent plate
After the 48 ± 2 hours incubation period, the presence of precipitate was determined in each well by microscopic inspection.
- Luminescence flash signal to evaluate induction signal - white plates
After incubation, the supernatants from the white assay plates were discarded. The cells were washed once with D-PBS. A volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 ± 2 minutes at room temperature and under orbital shaking. The plates containing the passive lysis buffer were then placed in the luminometer for reading. 50 μL of the luciferase substrate was added to each well, 1 second after this addition, the luciferase signal was integrated for 2 seconds. Thus, the cycle time to read one plate was approximately 10 min.
-Absorbance signal to evaluate the cytotoxicity - transparent plate
For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium. A volume of 27 μL of MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate. The plates were covered with a sealing membrane and returned at 37 °C in the incubator in humidified atmosphere for 4 hours (± 10 minutes). At the end of the incubation period, the medium was removed and a volume of 200 μL of a 10 % SDS solution was added to each well. The plates were covered with a sealing membrane and placed at 37 °C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells. After the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

ANALYSIS OF RESULTS
-Data evaluation was performed using a validated Excel sheet. The generated raw data (luminescence data for the luciferase activity and absorbance data for the MTT test) were pasted into an Excel template, and all data processing was performed automatically.
-For the MTT and the luciferase data, the background value recorded in the empty well without cells (blank) was subtracted. For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells. For the luciferase data, the average value of the six negative control wells was set to one and for each well in the plate, the fold induction was calculated in relation to this value.
-For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:
. Imax: maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured.
. EC1.5: concentration at which a 1.5-fold luciferase gene induction is obtained.
. IC50 and IC30: concentrations effecting a reduction of cellular viability by 50 % and 30 %.
. Indication whether significant 1.5-fold gene induction occurred below the IC30.
The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose-response curves or large variation may lead to wrong extrapolations.
Also, the individual and overall geometric means IC50 and IC30 were calculated, when applicable.

ACCEPTANCE CRITERIA
Each run was considered valid if the following criteria were met:
- The positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations.
- The average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between two and eight. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferase activity at increasing concentrations for the positive control.
- The average Coefficient of Variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20 %.

EVALUATION CRITERIA
The results of each run are analysed individually and if the test material is classified as positive in two runs, the final outcome is considered positive. If the test material is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.
The test material is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test).
- At the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70 %.
. The EC1.5 value is < 1000 μM (or < 200 μg/mL for test material without MW).
. There is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).

Positive control results:

The results for the positive control were Imax = 10.66 and EC1.5 = 9.50 µm (run 1) and Imax = 6.00 and EC1.5 = 9.41 µm (run 2). These values fall within the historical data ranges of Imax < 11.2 and EC1.5 = 0.8 to 27.1
All acceptance criteria were met for the positive and negative controls, both runs were therefore considered to be valid.

Key result

Run / experiment:

other: Geometric mean

Parameter:

other: IC30

Value:

8.61

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

value in µm

Key result

Run / experiment:

other: Geometric mean

Parameter:

other: IC50

Value:

9.52

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

value in µm

Run / experiment:

other: 1

Parameter:

other: EC1.5

Value:

1.32

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

value in µm

Run / experiment:

other: 2

Parameter:

other: EC1.5

Value:

2.96

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

value in µm

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

22.97

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

14.31

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS: KERATINOSENS RUN
First run
-All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
-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 of 10.66). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.
This run was 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:
. A slight to strong precipitate was observed in treated wells at concentrations ≥ 7.81 μM,
. A decrease in cell viability (i.e. cell viability < 70 %) was noted at concentrations ≥ 7.81 μM, the corresponding IC30 and IC50 were calculated to be 6.19 and 6.82 μM, respectively,
. Statistically significant gene-fold inductions above the threshold of 1.5 were noted at the concentrations of 1.95, 3.91 and 7.81 μM with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations,
. The Imax was 22.97 and the calculated EC1.5 was 1.32 μM.
Thus, the criteria of a positive response were met.
 
Second run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
This run was performed using the following concentrations: 0.46, 0.64, 0.91, 1.28, 1.81, 2.55, 3.59, 5.06, 7.13, 10.1, 14.2 and 20 μM in culture medium containing 1 % DMSO.
At these tested concentrations:
. A slight to strong precipitate was observed in treated wells at all tested concentrations,
. A decrease in cell viability (i.e. cell viability < 70 %) was noted at concentrations ≥ 14.2 μM, the corresponding IC30 and IC50 were calculated to be 11.98 and 13.29 μM, respectively,
. Statistically significant gene-fold inductions above the threshold of 1.5 were noted at concentrations ≥ 3.59 μM with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations,
. The Imax was 14.31 and the calculated EC1.5 was 2.96 μM.
Thus, the criteria of a positive response were met.

Global analysis from both runs
The geometric means IC30 and IC50 of the two validated runs were calculated to be 8.61 and 9.52 μM, respectively.
The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive.
This positive 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 skin sensitisation potential.

SOLUBILITY TEST
The test material was found to be soluble in DMSO at 200 mM, therefore this was selected for the preparation of the test material stock formulations.
A strong precipitate was observed once the test material stock formulation was diluted in the treatment culture medium to a final concentration of 2000 μM.

ACCEPTANCE OF RESULTS:
All acceptance criteria were met for the positive and negative controls, both runs were therefore considered to be valid.

Table 1: Summary of results

	Imax	EC1.5	IC50	IC30
Mean	18.64	n.r.	n.r.	n.r.
Geometric mean	n.r.	1.97	9.52	8.61
SD	6.12	1.16	4.57	4.09

n.r. = not requested by the OECD guideline

 

Interpretation of results:

other: Classification will be determined in conjuction with other sensitisation studies as part of an integrated testing strategy.

Conclusions:

Under the conditions of this study, the test material positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor. This positive 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 skin sensitisation potential.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442D, using the Keratinosens assay under GLP conditions.

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 material 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 material was dissolved in DMSO at 200 mM.

All acceptance criteria were met for the positive and negative controls, both runs were therefore considered to be valid.

First Run:

At the tested concentrations, a slight to strong precipitate was observed in treated wells at concentrations ≥ 7.81 μM. A decrease in cell viability (i.e.cell viability < 70 %) was noted at concentrations ≥ 7.81 μM; the corresponding IC30 and IC50 were calculated to be 6.19 and 6.82 μM, respectively. Statistically significant gene-fold inductions above the threshold of 1.5 were noted at the concentrations of 1.95, 3.91 and 7.81 μM with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations.The Imax was 22.97 and the calculated EC1.5 was 1.32 μM. Thus, the criteria of a positive response were met.

Second run:

At the tested concentrations, a slight to strong precipitate was observed in treated wells at all tested concentrations. A decrease in cell viability (i.e.cell viability < 70 %) was noted at concentrations ≥ 14.2 μM. The corresponding IC30 and IC50 were calculated to be 11.98 and 13.29 μM, respectively. Statistically significant gene-fold inductions above the threshold of 1.5 were noted at concentrations ≥ 3.59 μM with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations. The Imax was 14.31 and the calculated EC1.5 was 2.96 μM.

Thus, the criteria of a positive response were met.

Global analysis from both runs:

The geometric means IC30 and IC50 of the two validated runs were calculated to be 8.61 and 9.52 μM, respectively. The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive 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 skin sensitisation potential.

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and was considered to activate the Nrf2 transcription factor.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Chavallier 2017

The potential of the test material to act as a sensitiser was investigated in a GLP study in accordance with the standardised guidelines OECD 442C. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

This test was conducted as part of a tiered strategy for skin sensitization assessment. 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 based on 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 test item was dissolved at 100 mM in acetone. 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 (% interference < 10 %). As a result, the mean percent depletion values were calculated for each peptide using the formula described in Data analysis and calculation. For the cysteine peptide, the mean depletion value was 29.59 % and for the lysine peptide, the mean depletion value was 0.00 %.

The mean of the percent cysteine and percent lysine depletions was equal to 14.80 %. However, since precipitates were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated.

Since the mean of the percent cysteine and percent lysine depletions was between 6.38 and 22.62 %, the test item was considered to have a low peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization, though with limitations due to test item precipitation with the peptides.

Under the experimental conditions of this study, the test item was considered to have a low peptide reactivity, though with limitations due to test item precipitation with the peptides. The test item is considered positive in the DPRA assay.

Michel (2017)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442D, using the Keratinosens assay under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 material 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 material was dissolved in DMSO at 200 mM.

All acceptance criteria were met for the positive and negative controls, both runs were therefore considered to be valid.

First Run:

At the tested concentrations, a slight to strong precipitate was observed in treated wells at concentrations ≥ 7.81 μM. A decrease in cell viability (i.e.cell viability < 70 %) was noted at concentrations ≥ 7.81 μM; the corresponding IC30 and IC50 were calculated to be 6.19 and 6.82 μM, respectively. Statistically significant gene-fold inductions above the threshold of 1.5 were noted at the concentrations of 1.95, 3.91 and 7.81 μM with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations.The Imax was 22.97 and the calculated EC1.5 was 1.32 μM. Thus, the criteria of a positive response were met.

Second run:

At the tested concentrations, a slight to strong precipitate was observed in treated wells at all tested concentrations. A decrease in cell viability (i.e.cell viability < 70 %) was noted at concentrations ≥ 14.2 μM. The corresponding IC30 and IC50 were calculated to be 11.98 and 13.29 μM, respectively. Statistically significant gene-fold inductions above the threshold of 1.5 were noted at concentrations ≥ 3.59 μM with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations. The Imax was 14.31 and the calculated EC1.5 was 2.96 μM.

Thus, the criteria of a positive response were met.

Global analysis from both runs:

The geometric means IC30 and IC50 of the two validated runs were calculated to be 8.61 and 9.52 μM, respectively. The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive 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 skin sensitisation potential.

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and was considered to activate the Nrf2 transcription factor.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification for skin sensitisation.