[No public or meaningful name is available]

Endpoint summary

Description of key information

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. In this context three studies were conducted to evaluate skin sensitising potential of the test item:

-OECD 442C (Direct Peptide Reactivity Assay (DPRA))

-OECD 442D (ARE-Nrf2 Luciferase Test Method (KeratinoSens™))

-OECD 442E (human Cell Line Activation Test (h-CLAT))

 

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:

weight of evidence

Study period:

May 04, 2022 to July 28, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

adopted 30 June 2022

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Justification for non-LLNA method:

The correlation of upregulation of immunologically relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event of the skin sensitisation process as described by the AOP for skin sensitisation. This method, which measures the markers of DC activation, using the DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals. Therefore, this test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues. Depending on the regulatory framework, positive results generated with these methods may be used on their own to classify a chemical into UN GHS category 1.

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Preparation of the Test Item

The test item was freshly prepared immediately prior to use.
The test item was dissolved in 0.9% NaCl solution at a concentration of 500 mg/mL. Vortex mixing was used to aid solubilisation. No correction factor for the purity was used.
The working stock solutions were prepared by diluting each stock solution 50 times with cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (0.9% NaCl solution) was present at a constant volume ratio of 1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Test System

FACS
FACS: BD FACSLyric
Software: BD FACSuite 1.2.1

Cell line
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 106 cells/mL.
Cells were cultured in 175 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 µg/mL streptomycin in a humidified incubator at 37 +- 1°C and 5% CO2.

Dose Groups
1. Medium Control: cell culture medium
2. Solvent Control: cell culture medium
0.2% DMSO (v/v) in cell culture medium
3. Positive Control: 4 µg/mL DNCB
4. Test Item: 8 concentrations of the test item
(dose finding assay/ main experiment)
dose finding assay 1:
5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 µg/mL
main experiment 1 and 2:
5000.00, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49 and 1395.41 µg/mL

Pre-Experiments

Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB CAS No. 97-00-7, ≥ 99% purity) at a final concentration of 4 µg/mL and nickel sulphate (CAS No. 10101-97-0, 99% purity) at a final concentration of 100 µg/mL served as positive control while lactic acid (CAS No. 50-21-5, ≥ 99% purity) at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

Solvent Finding
Solubility of the test item was determined prior to the dose-finding assay experiment. The test item was soluble in 0.9% NaCl at a final concentration of 500 mg/mL.

Experimental Procedure

Dose Finding Assay
Starting from 500 mg/mL solutions of the test chemical, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 50-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution
For testing, THP-1 cells were pre-cultured for 48 h -72 h in culture flasks at a cell density of
0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well).
The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at
37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer.
200 µL of the cell suspension was transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL.
The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration.
The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation utilizing equation 10 2. The CV75 value was used to calculate the concentration range of the test item for the main experiment.
 
10.7.2. CD54 and CD86 Expression
The test item was dissolved using 0.9% NaCl as determined in the pre-experiment. As the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item (500 mg/mL) was used as starting dose.
The test item was diluted to a concentration of 500 mg/mL and then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 50-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for 48 h – 72 in culture flasks at a cell density of
0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 µL of the cell suspension was seeded into a 24 well flat-bottom plate (1 x 106 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at
37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer in an appropriate manner. After washing with FACS buffer twice, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. The cell viability was calculated.

Data Analysis
FACS data analysis was performed using the software BD FACSuite 1.2.1. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 (EC150) and Effective Concentration 200 (EC200) values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands.


Evaluation of the Results
Prediction Model
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9% NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.
A negative result for test items with a Log KOW > 3.5 should be considered as inconclusive.

Vehicle / solvent control:

other: Since the test item was solubilized in either cell culture medium or 0.9% NaCl, the medium control served as solvent control. Since the positive control was solubilized in DMSO, a DMSO control was included and served as solvent control for the PC

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86.

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD86>200 [442E]

Value:

0 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 95.3% (CD86).

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD86>200 [442E]

Value:

0 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 95.7% (CD86)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD54>150 [442E]

Value:

0 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 95.2%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD54>150 [442E]

Value:

0 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 92.8% (CD54).

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experimental runs. Therefore, the test item can be considered not to activate dendritic cells, the third key event in the skin-sensitization AOP.
The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of Integrated Approach to Testing and Assessment (IATA) in combination with other complementary information e.g. derived from in vitro assays addressing other key events of the skin-sensitisation AOP.

Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study, the test item was dissolved in 0.9% NaCl. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL (final concentration in the test media 5000 µg/mL to 39.06 µg/mL) were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

5000, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49 and 1395.41 µg/mL

In all experiments no precipitation and no turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 95.7% (CD86), 92.8% (CD54) and 94.6% (isotype IgG1 control) in the first experiment and to 95.3% (CD86), 95.2% (CD54) and 92.9% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Therefore, under the conditions of this study the test item was negative for dendritic cell activation.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 26, 2022 to May 13, 2022

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 KeratinoSens™ test method)

Version / remarks:

adopted: June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line (e.g. KeratinoSens™) derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Therefore, this test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues. Depending on the regulatory framework, positive results generated with these methods may be used on their own to classify a chemical into UN GHS category 1.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

Preparation of the test item
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; AppliChem; Lot No.: 0001926479). A stock solution of 200 mM was prepared by pre-weighing the test material into a suitable tube. A factor of 1.14 to correct for the purity of the test item was used.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Dose Groups
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per 96-well plate in every independent run.


Experimental Procedure
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 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.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37°C ± 1 °C and 5% CO2.

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were 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 were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 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 °C ± 1 °C and 5% CO2 overnight (experiment 2) or over the weekend (experiment 1). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Vehicle / solvent control:

cell culture medium

Negative control:

other: DMSO (AppliChem; Lot No.: 0001926479) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate.

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The controls confirmed the validity of the study .

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in two independent experiment runs. Therefore, the test item can be considered not to activate the antioxidant response element (ARE)-dependent pathway in keratinocytes, the second key event in the skin sensitization AOP.
The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approaches such as IATA, in combination with other complementary information e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study, the test item was dissolved in DMSO.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. The following concentration range was tested in the assay:

2000.00, 1000.00, 500.00, 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered not to activate the antioxidant response element (ARE)-dependent pathway in keratinocytes, the second key event in the skin sensitization AOP.

The controls confirmed the validity of the study.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 26, 2022 to February 24, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

adopted June 14, 2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.

This test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into UN GHS “Category 1”.
Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Incubation of the Test Item with the Cysteine and Lysine Peptide

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. Samples were prepared according to the scheme described in Table 1

Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 400x g) to force precipitates to the bottom of the vial.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.

Preparation of the HPLC Standard Calibration Curve

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 indicated in the following:
- STD1: 0.534
- STD2: 0.267
- STD3: 0.134
- STD4: 0.067
- STD5: 0.033
- STD6: 0.017
- STD7: 0.000

HPLC Preparation and Analysis

Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days, all test chemical solutions were freshly prepared for both assays on each day.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

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 67.31%.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

0.22 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as a “non-sensitiser”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) 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 373.323 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 no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_Acetonitril).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was £ 6.38% (0.11%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Based on the available data, the substance does not need to be classified and labelled according to Regulation (EC) No.1272/2008 (CLP).