Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12-16 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

EC Number:

275-085-2

EC Name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Cas Number:

70983-43-6

Molecular formula:

see structural formula below, for n=4 C13H24N2O3 HO.Na

IUPAC Name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Test material form:

liquid

Remarks:

oily liquid

Details on test material:

Batch No.: 54542L16
Storage at room temperature, protected from light.

In vitro test system

Details on the study design:

EXPERIMENTAL PROCEDURES

DOSE FORMULATION: Preparation of Test Item
For preparation of the 100 mM test item solution, a single purity of 99.99% was used which was determined by the sum of the proportion of its constituents (excluding water), and a single apparent molecular weight of 282.475 g/mol had been determined by considering the individual molecular weights of each component in the mixture (excluding water) and their individual proportions. The resulting purity and apparent molecular weight were used to calculate the weight of test chemical necessary to prepare a 100 mM solution.
For both the cysteine and lysine reactivity assay 52.98 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1876 µL ACN after vortex mixing and 1 minute of sonication to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting
the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

PEPTIDES:
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.

BUFFERS USED:
- Phosphate buffer: ca 100 mM, pH 7.5.
- Ammonium acetate buffer: ca 100 mM, pH 10.2.

PREPARATION PEPTIDE STOCK SOLUTIONS:
- CYSTEINE: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- LYSINE: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

REFERENCE CONTROL SOLUTIONS
- CYSTEINE:Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.
- LYSINE: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN

SAMPLE INCUBATION;
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

HPLC-PDA ANALYSIS
SPCC and SPCL peak areas in the samples were measured by HPLC PDA. Sample analysis was performed using the following system:
-System 1 (used for Cysteine Reactivity Assay):
-Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
-MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
-LC Column oven 300 (Thermo Scientific)
-Surveyor PDA detector (Thermo Scientific)
-System 2 (used for Lysine Reactivity Assay):
-Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
-HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
-Column Oven #151006 (Grace, Worms, Germany)
Surveyor PDA detector (Thermo Scientific)
All samples were analyzed according to the HPLC-PDA method presented in Table 1 (Appendix 1). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 (Appendix 1).

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
-The standard calibration curve had to have an r2>0.99.
-The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
-The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion
-The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
-The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
- The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
-The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM

ANALYSIS -DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= [1-((Peptide Peak Area in Replicate Injection (at 220 nm))/(Mean Peptide Peak Area in Reference Controls (at 220 nm)))]×100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
ANALYSIS-DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see table below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
Cysteine 1:10 / Lysine 1:50 Prediction Model

0% = Mean % depletion = 6.38% No or minimal reactivity Negative
6.38% < Mean % depletion = 22.62% Low reactivity Positive
22.62% < Mean % depletion = 42.47% Moderate reactivity Positive
42.47% < Mean % depletion = 100% High reactivity Positive

Results and discussion

Positive control results:

The mean depletion value for the positive control was 60.8% showing a high reactivity (Sensitizer)
The mean Percent SPCCysteine Depletion for the positive control cinnamic aldehyde was 75.3%
The mean Percent SPCLysine Depletion for the positive control cinnamic aldehyde was 54.3%

In vitro / in chemico

Results

Key result

Run / experiment:

other: DPRA cysteine and lysine prediction model

Parameter:

other: % Mean of SPCC and SPCL depletion

Value:

0.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Cinnamic aldehyde

Remarks on result:

other:

Remarks:

Negative: No or minimal reactivity

Other effects / acceptance of results:

The co-elution control (CC) as well as the test item samples were visually inspected. No precipitate was observed in any of the samples.

SYSTEM SUITABILITY FOR THE CYSTEINE ASSAY
The correlation coefficient (r2) of the SPCC standard calibration curve was 0.996. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.495 ± 0.009 mM while the mean peptide concentration of Reference Controls C was 0.491 ± 0.006 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 0.9%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 17.90. The mean A220/A258 ratio ± 10% range was 16.11-19.69. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 75.3% ± 1.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Results Cysteine Reactivity Assay for the Test Item
In the CC sample a peak was observed at the retention time of SPCC. This indicated that there was co-elution of the test item with SPCC. For the 209012/A-cys samples, the mean SPCC A220/A258 area ratio was 6.99. Since this was outside the 16.11-19.69 range, this again indicated that there was co-elution of the test item with SPCC.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 1.6% ± 0.5%, however, since co-elution of the test item with SPCC has occurred, this result has to be interpreted with due care. The peak observed at a wavelength of 220 nm at the retention time of SPCC in the chromatogram of the CC sample equals to 6.6% of the averaged peak area of the SPCC peak
in the corresponding chromatograms of the 209012/A-cys samples. Consequently, the percentage of SPCC depletion might be underestimated.

SYSTEM SUITABILITY FOR THE LYSINE ASSAY
The correlation coefficient (r2) of the SPCL standard calibration curve was 0.998. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.489 ± 0.007 mM while the mean peptide concentration of Reference Controls C was 0.481 ± 0.005 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
The CV of the peptide areas for the nine Reference Controls B and C was 2.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time
The mean area ratio (A220/A258) of the Reference Control samples was 15.05. The mean A220/A258 ratio ± 10% range was 13.55-16.56. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
.
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 54.3% ± 3.0%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for the Test Item
Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. No precipitate was observed in any of
the samples.
In the CC sample a peak was observed at the retention time of SPCL. This indicated that there was co-elution of the test item with SPCL. For the 209012/A-lys samples, the mean SPCL A220/A258 area ratio was 12.69. Since this was outside the 13.55-16.56 range, this again indicated that there was co-elution of the test item with SPCL.
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Item was 0.0% ± 0.0%, however, since co-elution of the test item with SPCL has occurred, this result has to be interpreted with due care. The peak observed at a wavelength of 220 nm at the retention time of SPCL in the chromatogram of the CC sample equals to 6.4% of the averaged peak area of the SPCL peak
in the corresponding chromatograms of the 209012/A-lys samples. Consequently, the percentage of SPCL depletion might be underestimated.

Any other information on results incl. tables

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forImidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and     reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts	1.6%	±0.5%	0.0%	±0.0%	0.8%	Negative: No or minimal reactivity

SD = Standard Deviation.

Using the cysteine and lysine prediction model (see Table below) the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Cysteine 1:10 / Lysine 1:50 Prediction Model

Mean of cysteine and lysine % depletion	Reactivity class	DPRA prediction
0% = Mean % depletion = 6.38%	No or minimal reactivity	Negative
6.38% < Mean % depletion = 22.62%	Low reactivity	Positive
22.62% < Mean % depletion = 42.47%	Moderate reactivity
42.47% < Mean % depletion = 100%	High reactivity

Applicant's summary and conclusion

Interpretation of results:

other: no or minimal reactivity class

Remarks:

Study will be used for classificatin in combination with other studies (Weight of Evidence)

Conclusions:

In conclusion, this DPRA test is valid. Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts (CAS Nr. 70983-43-6) was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since in both the SPCC and SPCL incubations co-elution of the test item with the peptide has occurred, the percentages of SPCC and SPCL depletion might be underestimated. Therefore, this negative result is uncertain and has to be interpreted with due care.

Executive summary:

The objective of this study was to determine the reactivity of Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts (CAS Nr. 70983-43-6) towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures described in this report were based on the most recent OECD guideline 442C (04 February 2015).

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of theDirect Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.996	>0.99	0.998
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.495 ± 0.009	0.50 ± 0.05	0.489 ± 0.007
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.491 ± 0.006	0.50 ± 0.05	0.481 ± 0.005
CV (%) for RC samples B and C	<15.0	0.9	<15.0	2.2
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	75.3	40.2-69.0	54.3
SD of peptide depletion cinnamic aldehyde (%)	<14.9	1.0	<11.6	3.0
SD of peptide depletion for the test item (%)	<14.9	0.5	<11.6	0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate was observed in any of the samples. 

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 1.6% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.8% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forImidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and     reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts	1.6%	±0.5%	0.0%	±0.0%	0.8%	Negative: No or minimal reactivity

SD = Standard Deviation.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts (CAS Nr. 70983-43-6) was negative in the DPRA and was classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since in both the SPCC and SPCL incubations co-elution of the test item with the peptide has occurred, the percentages of SPCC and SPCL depletion might be underestimated. Therefore, this negative result is uncertain and has to be interpreted with due care.