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

Endpoint summary

Description of key information

-       DPRA (OECD 442C): mean peptide depletion: 0.8%, minimal reactivity, Non sensitizer.

-       Keratinosens^TM(OECD 442D): Negative, Non sensitizer

Cytotoxic: IC₃₀ 135 µg/mLand 82 µg/mL; and IC₅₀190 µg/mL and 127 µg/mL

Imax: 1.15 and 1.28 fold induction

-       H-CLAT (OECD 442E): Sensitizer. Minimum induction threshold (MIT) of 43 µg/mL.

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:

12-16 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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

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%

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.

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4-15 December 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

Details on the study design:

- Test concentrations: final test concentrations of 400, 200, 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39 and 0.20 µg/mL
- All concentrations of the test item were tested in triplicate.
- Positive control: Ethylene dimethacrylate glycol, final concentration 7.8 to 250 µM (final concentration DMSO of 1%).
- Negative control: vehicle: Milli-Q water (Millipore Corp., Bedford, MA., USA), 1% DMSO in exposure medium (for positive controls)

- Test System A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland).

- Cell culture:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Manteinance Medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

- Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 – 100 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.9 – 36.6°C).

EXPEIMENTAL DESIGN
- Plating of cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each test item, one plate was used for the luciferase activity measurements, and one parallel plate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+8 in experiment 1 and P+10 in experiment 2.
- Treatment of cells
The medium was removed and replaced with fresh culture medium (150 µL culture medium containing serum but without Geneticin) to which 50 µL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0 °C in the presence of 5% CO2. In total 2 experiments were performed.

- Luciferase activity measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).
- Cytotoxicity assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 µM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- Data analysis
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

- Data interpretation
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 µM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Positive control results:

• Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.38 and the EC1.5 95 µM.
• Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.75 and the EC1.5 51 µM.

Key result

Run / experiment:

other: 1

Parameter:

other: maximal average fold induction of luciferase activity (Imax)

Value:

1.15

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax:2.38

Key result

Run / experiment:

other: 2

Parameter:

other: maximal average fold induction of luciferase activity (Imax)

Value:

1.28

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Imax: 2.75

Key result

Run / experiment:

other: 1

Parameter:

other: EC 1.5 (µM) (concentration for which induction of luciferase activity is above the 1.5 fold threshold)

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5: 95 µM

Remarks on result:

no indication of skin sensitisation

Remarks:

no EC1.5 could be calculated

Key result

Run / experiment:

other: 2

Parameter:

other: EC 1.5 (µM) (concentration for which induction of luciferase activity is above the 1.5 fold threshold)

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5: 51 µM

Remarks on result:

no indication of skin sensitisation

Remarks:

no EC1.5 could be calculated

Other effects / acceptance of results:

The test Item showed toxicity. The calculated IC30 was 135 µg/mL and the calculated IC50 was 190 µg/mL for the first run.
The calculated IC30 was 82 µg/mL and the calculated IC50 was 127 µg/mL for the second run.

Acceptance criteria:
•The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
•The EC1.5 of the positive control was between 5 and 125 µM (95 µM and 51 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.38-fold and 2.75-fold in experiment 1 and 2, respectively).
•The average coefficient of variation of the luminescence reading for the negative (solvent) control Milli-Q water was below 20% (8.0% and 8.2% in experiment 1 and 2, respectively).
•Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (7.6% and 6.1% in experiment 1 and 2, respectively).

Table1          
Overview Luminescence Induction and Cell Viability of the Test Item in Experiment 1 and 2

Concentration (µg/mL)	0.20	0.4	0.8	1.6	3	6	13	25	50	100	200	400
Exp 1 luminescence	1.00	1.06	1.14	1.11	1.15	1.15	1.06	1.04	0.88	0.63	0.58	0.00
Exp 1 viability (%)	110.7	105.7	97.2	94.7	91.2	85.7	95.7	90.5	99.6	82.8	46.3	-0.1
Exp 2 luminescence	0.96	1.14	1.18	1.23	1.28	1.17	0.95	0.96	0.84	0.68	0.49	0.00
Exp 2 viability (%)	101.3	125.6	106.2	94.1	102.6	98.0	97.1	98.2	93.7	56.4	33.0	-0.1

 

Table2          
Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	0.96	1.01	1.24	1.36	1.63***	2.38***
Exp 1 viability (%)	103.9	102.9	100.9	106.2	110.0	114.6
Exp 2 luminescence	1.12	1.24	1.29	1.63***	1.88***	2.75***
Exp 2 viability (%)	110.2	120.2	128.4	129.9	113.9	121.5

^***p<0.001 Student’s t test

 

Table3          
Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5(µg/mL)	Imax	IC30(µg/mL)	IC50(µg/mL)
Test item Experiment 1	NA	1.15	135	190
Test item Experiment 2	NA	1.28	82	127
	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Pos Control Experiment 1	95	2.38	NA	NA
Pos Control Experiment 2	51	2.75	NA	NA

NA = Not applicable

Interpretation of results:

other: the Test item did not induce activation of the ARE-dependant pathway in keratinocytes

Remarks:

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

Conclusions:

In conclusion, Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3- (hydroxyethyl), hydroxides, sodium salts (CAS No. 70983-43-6) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3- (hydroxyethyl), hydroxides, sodium salts (CAS No. 70983-43-6) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 54542L16 of the test item was an orange oily liquid. The test item was suspended in Milli-Q water at 40 mg/mL. At concentrations of 5 mg/mL and higher the test item formed a suspension in Milli-Q water whereas at 2.5 mg/mL and lower it was fully soluble. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.20 – 400 µg/mL (2-fold dilution series). The highest test concentration was thehighest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

·       The luciferase activity induction obtained with the positive control,Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 

·       The EC_1.5of the positive control was between 5 and 125 µM (95 µM and 51 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.38-fold and 2.75-fold in experiment 1 and 2, respectively).

·       The average coefficient of variation of the luminescence reading for the negative (solvent) control Milli-Q water was below 20% (8.0% and 8.2% in experiment 1 and 2, respectively).

·       Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (7.6% and 6.1% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

The test item showed toxicity (IC₃₀values of 135µg/mLand 82µg/mLand IC₅₀values of 190µg/mL and 127 µg/mLin experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.15-fold and 1.28-fold in experiment 1 and 2 respectively. The test itemis classified as negative in the KeratinoSens^TMassaysince negative results (<1.5-fold induction) were observed at test concentrations = 200µg/mL.

In conclusion, Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3- (hydroxyethyl), hydroxides, sodium salts (CAS No. 70983-43-6) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 December 2017 - 15 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals Nr. 442E, In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), 9 October 2017

Deviations:

yes

Remarks:

See details on study design section.

Qualifier:

according to guideline

Guideline:

other: DB-ALM (INVITTOX) (2014) Protocol 158: human Cell Line Activation Test (h-CLAT),

Deviations:

not specified

Principles of method if other than guideline:

The 2-mercaptoethanol was not used in the culture medium because this toxic compound is not necessary for the culture of THP-1.
For citotoxicity testing4-fold dilution factor instead 2-fold was used because, a significant cytotoxicity for the test item (surfactant) was expected.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

PRINCIPLE OF THE TEST:

The h-CLAT method is proposed to address the third key event (DC activation) of the skin sensitisation AOP by quantifying changes in the expression of cell surface markers associated with the process of activation of DC (i.e. CD86 and CD54), in the human monocytic leukaemia cell line THP-1, following exposure to sensitizers. The measured expression levels of CD86 and CD54 cell surface markers are then used for supporting the discrimination between skin sensitizers and non-sensitizers.
THP-1 cell cultures are exposed to the test item for 24 hours. After exposure, the expression of two cell surface antigens, CD86 and CD54, was measured by flow cytometry method. THP-1 cells show upregulation of CD86 or CD54 expression, known as dendritic cell activation, following treatment with various sensitizers, but not non-sensitizers.

Criteria for prediction as positive:
Two or three independent experiments, at any dose should exceed the positive criteria: CD86 = 150% or CD54 = 200% of their respective vehicle control

TEST SYSTEM
THP-1 (monocytic leukeamia cell line) cells provided from American Type Culture Collection (batch 61077351).
Cells were stored in liquid nitrogen and the assays were performed thanks to a master bank (batch THP101008) supplied by Biopredic International (Saint Grégoire – France). Cryopreserved cells were thawed. Cells were cultured, at 37°C under 5% CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10% Fœtal Calf Serum, 100 units/mL penicillin and 100 µg/mL streptomycin. THP-1 were routinely seeded every 3-4 days at the density of 0.15 to 0.2 x 106 cells/mL.
For testing, THP-1 cells were seeded at a density of 0.2 x 106 and pre-cultured in culture flasks for 72 hours.
Control of cells: The quality of each batch of THP-1 cells was checked. Viability of the cells must be above 90%.

Positive control substances: 2,4-Dinitrochlorobenzene (DNCB) and NiSO4
Negative control substance: Lactic acid (LA)

PRELIMINARY STUDY: CYTOTOXICITY ASSAY
The cytotoxicity of the test item was evaluated in order to select at least 4-5 concentrations able to induce cytotoxicity, around 50%, for the highest one. Assessment of cell toxicity was performed by determining cell viability on THP-1 cells, using the 7-AAD inclusion methods.
Eight concentrations of test item were prepared by a four-fold serial dilution from a maximum final concentration of 1000 µg/mL or lower, depending on its solubility limit.
In the day of testing, cells harvested from culture flask were suspended with fresh culture medium at
2 x 106 cells/mL. Then, THP-1 cells were distributed into a 24 well flat-bottom plate with 500 µL
(1 x 106 cells/well) with various concentrations of test item (1:1 ratio) for 24±0.5 hours at 37 °C under 5 % CO2. After treatment cells were washed twice with phosphate-buffered containing 0.1% (w/v) bovine serum albumin identified as FACS buffer (Fb). The cells were stained with 7-AAD (5 µg/mL final concentration). Then cells were analysed with flow cytometry using GUAVA (Merck Millipore, France) and InCyte software to measure cell viability. The living cells (7-AAD-) gate was set in the 7-AAD negative area. 104 7-AAD- cells were counted as the living population.
In case of product cytotoxicity, the CV75 value was calculated by log-linear interpolation using the following equation:

Log CV75 = (75-c) x Log (b) – (75-a) x Log (d)/a – c

a: minimum value of cell viability over 75%
c: maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively
According to the results the dose levels for the main study were selected.

MAIN STUDY: ACTIVATION TESTS
Each activation test was performed on eight concentrations.
THP-1 cells were plated at 1*106 cells/mL/well in 24 well plates and treated for 24±0.5 hours with selected test item concentrations. After treatment cells were washed twice with Fb. Then cells were stained for 30 min at 4°C with the following fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAbs): anti-human CD54, anti-human CD86; FITC labelled-mouse IgG1. Using the manufacturer’s recommended dilutions, cells were incubated with above mAbs at 6 µL/3*105 cells /50µL for the anti-human CD86 mAb, and 3 µL/3*105 cells /50µL for the anti-human CD54 mAb. FITC labelled-mouse IgG1 was used as an isotype control at a dilution of 3 µL/3*105 cells /50µL. Then, the cells were stained also with 7-AAD for 30 min at 4°C. After washing and resuspension with Fb, the fluorescence intensities of the THP-1 cell surface markers were then analysed by flow cytometry using GUAVA (Merck Millipore, France) and InCyte software, on 10000 living cells.

DATA ANALYSIS AND INTERPRETATION:
The Relative Fluorescence Intensity (RFI) is used as an indicator of CD86 and CD54 expression. RFI is calculated with the following formula:

RFI = (MFI of test item-treated cells – MFI of test item-treated isotype control cells)/(MFI of vehicle control cells – MFI of vehicle isotype control cells)

When the cell viability was less than 50%, RFI was not calculated because of the diffuse labelling of cytoplasmic structures that are generated following cell membrane destruction.

ACTIVATION TEST POSITIVE CRITERIA:
RFI of CD54 = 200% and RFI of CD86 = 150% of their respective control at any tested concentration.

ACCEPTABILITY CRITERIA:
This study is considered valid if the following criteria are fully met:
- In the positive control (DNCB):
RFI values of both CD86 and CD54 should be over the positive criteria (CD54 = 200% and CD86 = 150%).
The cell viability should be more than 50%

- In the vehicle control (medium, 0.9% NaCl, DMSO, Ethanol etc.):
Cell viability should be more than 90%
In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 = 150% and CD54 = 200%) compared to the medium control
The MFI ratio of both CD86 and CD54 to isotype control should be > 105%

- In the test item:
Cell viability should be more than 50% in at least four tested concentrations in each run.

PREDICTION MODEL:
If two or three independent experiments, at any dose exceed the positive criteria: CD86 = 150% or CD54 = 200% of their respective vehicle control, the chemical may be identified as a sensitizer. Otherwise it is identified as a non-sensitizer.

GUIDELINE DEVIATIONS:
- The 2-mercaptoethanol was not used in the culture medium because this toxic compound is not necessary for the culture of THP-1.
- The concentrations for cytotoxicity test were performed by a 4-fold serial dilution from the top concentration instead by a 2-fold serial, beacuse a significant cytotoxicity for the test item (surfactant) was expected.This had no impact on the quality of the results.

Positive control results:

NiSO4: Result CD54: RFI:5435
Result CD86: RFI: 232
Validity criteria: =200 (CD54) and =150 (CD86)

DNCB: Result CD54: RFI:600
Result CD86: RFI: 368
Validity criteria: =200 (CD54) and =150 (CD86)

DNCB and NiSO4 produced a positive response for both CD86 and CD54.

Key result

Run / experiment:

other: 2

Parameter:

other: EC200 (µg/mL)

Remarks:

concentration inducing 200% of CD54 RFI

Value:

53.6

Vehicle controls validity:

valid

Remarks:

CD54 RFI:100; CD86 RFI: 100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

2,4-Dinitrochlorobenzene CD54 RFI:600; CD86 RFI: 368

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 3

Parameter:

other: EC200 (µg/mL)

Remarks:

concentration inducing 200% of CD54 RFI

Value:

32.5

Vehicle controls validity:

valid

Remarks:

CD54 RFI:100; CD86 RFI: 100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

CD54 RFI:858; CD86 RFI: 345

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- No contamination was noticed during the study
- Quality control of the test system: all acceptability criteria were fulfilled (See Table 1 and 2) in the filed "Any other information on results"

CYTOTOXICITY ASSAY:
Cytotoxicity was induced on THP-1 cells by Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts. According to this cytotoxic profile, a CV75 value of 199 µg/mL could be determined. In the activation tests, therefore, the doses-range was chosen between 36.3 and 500 µg/mL to cover a cytotoxic profile until 50% of dead cells.

ACTIVATION TESTS:
Test system was validated as all acceptability criteria were fulfilled (Table 1 and 2).
Negative controls (RPMI) showed cell viability values acceptable regarding the acceptance criteria.
Positive controls showed an increase of CD54/86 expression (RFI = 200/150 respectively) compared to the negative control (Table 1, 2 and 3).
On the three activation experiments five to eight concentrations could be analysed.
In the first experiment, a dose increase was noticed for the CD54 expression with the test item at any tested non-cytotoxic concentrations. No increase of expression for CD86 marker was observed.
In the last two experiments, a dose-response relationship was noticed for CD54 marker with an increase of 2.27 to 11.06 fold of expression compared to the negative control. No increase of expression for CD86 marker was observed.

Negative control: Lactic acid produced a negative response for both markers.

Table1: CV and ratio of both markers CD54 and CD86 to isotype control obtained for vehicles and controls

Sample	CV (%)		Ratio CD54/IgG		Ratio CD86/IgG
	Criteria	Results	Criteria	Results	Criteria	Results
RPMI	>90%	98	>105	124	>105	214
0.9% NaCl	>90%	98	>105	122	>105	224
DMSO	>90%	98	>105	129	>105	217
NiSO4	>50%	80
DNCB	>50%	82
LA	>50%	97

Table 2: RFI values obtained for vehicles and controls

Sample	RFI
	Criteria	Results CD54	Results CD86
0.9% NaCl	=200 (CD54) and =150 (CD86)	98	113
DMSO	=200 (CD54) and =150 (CD86)	118	98
NiSO4	=200 (CD54) and =150 (CD86)	5435	232
2,4-Dinitrochlorobenzene	=200 (CD54) and =150 (CD86)	600	368
Lactic acid	=200 (CD54) and =150 (CD86)	148	79

Table 3: CD54/86 expression of the test item Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts after a 24-hour exposure period using the RFI.

Sample	Dose Level (µg/mL)	Experiment 1			Experiment 2			Experiment 3
		CD54	CD86	CV(%)	CD54	CD86	CV(%)	CD54	CD86	CV(%)
RPMI (Vehicle)	-	100	100	98	100	100	97	100	100	98
DMSO (solvent)	-	118	98	98	136	124	97	131	136	98
2,4 -Dinitrochlorobenzene (positive control)	4	600	368	82	725	312	82	858	345	84
Test item	36.3	ND	ND	ND	172	71	97	243	95	98
Test item	47.1	ND	ND	ND	200	75	97	257	86	98
Test item	61.3	ND	ND	ND	227	78	97	314	68	98
Test item	79.7	281	70	98	246	61	97	407	84	97
Test item	104	340	73	97	328	77	96	576	98	97
Test item	135	437	84	95	371	72	95	601	86	96
Test item	175	567	80	84	541	85	88	852	91	91
Test item	228	560	90	61	685	90	70	1106	122	70
Test item	296	491	78	26	ND	ND	ND	ND	ND	ND
Test item	385	146	228	3	ND	ND	ND	ND	ND	ND
Test item	600	62	251	3	ND	ND	ND	ND	ND	ND

Test item = Test item in vehicle

Based on linear regression and according to the guideline, the concentration inducing 200% of CD54 RFI (EC200, EC: Estimated Concentration) was calculated by extrapolation, for each experiment:

Experiment 2: EC200 = 53.6 µg/mL

Experiment 3: EC200 = 32.5 µg/mL

 

Then the average and the standard deviation were calculated from these individual values to obtain a final EC200.

Final EC200 = 43.0 ± 14.9 µg/mL

Interpretation of results:

other: positive dendritic cell activation (human monocytic leukaemia cell line THP-1) in vitro

Remarks:

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

Conclusions:

The test item Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts (CAS 70983-43-6) demonstrated an in vitro sensitizing potential with a Minimum Induction Threshold (MIT) of 43.0 µg/mL ± 14.9 µg/mL in condition of the experimental human Cell Line Activation Test, during this study.

Executive summary:

The objective of this study was to evaluate the in vitro intrinsic sensitizing potential of the test item Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts (CAS 70983-43-6). The method used for this study was the human cell line activation test (h-CLAT).

The h-CLAT method is proposed to address the third key event (DC activation) of the skin sensitisation AOP by quantifying changes in the expression of cell surface markers associated with the process of activation of DC (i.e. CD86 and CD54), in the human monocytic leukaemia cell line THP-1, following exposure to sensitizers. The measured expression levels of CD86 and CD54 cell surface markers are then used for supporting the discrimination between skin sensitizers and non-sensitizers.

Cytotoxicity was induced on THP-1 cells by Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts (CAS 70983-43-6). According to this cytotoxic profile, a CV75 value of 199 µg/mL could be determined.

In the activation tests, therefore, the dose-range was chosen between 36.3 and 500 µg/mL to cover a cytotoxic profile until 50% of dead cells.

In the assay conditions, a reproducible "increase" of the CD54 expression compared with the negative control at least for six dose-levels of Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts was noticed. No increase was observed for CD86 expression.

In the first experiment, a dose increase was noticed for the CD54 expression with the test item in all tested non-cytotoxic concentrations. No increase of expression for CD86 marker was observed.

In the last two experiments, a dose-response relationship was noticed for CD54 marker with an increase of 2.27 to 11.06 fold of expression compared to the negative control. No increase of expression for CD86 marker was observed.

Based on these results, the test item Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts demonstrated an in vitro sensitizing potential with a Minimum Induction Threshold (MIT) of 43.0 µg/mL under the conditions used during this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

-   QSAR DEREK: not performed as it is a complex UVCB substance

- A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in acetonitrile at 100 mM and incubated with the cysteine- and lysine-containing synthetic peptides. No precipitate was observed in any of the samples. The mean percent SPCC depletion for the test item was 1.6% ± 0.5% and the mean percent SPCL depletion was 0.0% ± 0.0%, however, since partial co-elution of the test item with SPCC and SPCL has occurred, these results have to be interpreted with due care. The peak observed at a wavelength of 220 nm at the retention time of SPCC and SPCL, respectively, in the chromatogram of the co-elution control sample equals only 6.6% of the averaged peak area of the SPCC peak and 6.4% of the averaged peak area of the SPCL peak in the corresponding chromatograms of the 209012/A-cys samples. For the SPCL peak, the value of 209012/A-lys-1, 2 or 3 minus the value of CClys-209012/A is still at or above the RClysC-1 value, which means still no depletion at all for lysine. Consequently, the percentage of SPCC depletion might be underestimated, but as the effect on the depletion peaks of SPCC/SPCL is only minimal, the mean SPCC depletion or mean of SPCC and SPCL depletion is not expected to be > 6.38%. Therefore, the result is considered to be negative when using the Cysteine 1:10/Lysine 1:50 prediction model.

- A valid Keratinosens assay was performed according to OECD 442D and GLP principles. The test item was suspended in Milli-Q water at 40 mg/mL. The 100-fold dilution of the 40 mg/mL Milli-Q water stock in DMEM-glutamax formed a clear solution (400 µg/mL). From this stock 11 spike solutions were prepared. The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in test concentrations of 0.20 – 400 µg/mL. No precipitate was observed at any dose level tested. Two independent experiments were performed.

The test item showed toxicity (IC30 values of 135 and 82 µg/mL and IC50 values of 190 and 127 µM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.15-fold and 1.28-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSens^TMassay since negative results (<1.5-fold induction) were observed at test concentrations up to 200 µg/mL. Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts (CAS No. 70983-43-6) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

- A valid h-CLAT assay was performed according to OECD 442E and GLP principles. Based on cell toxicity assays, in which cytotoxicity was observed with a CV75 of 199µg/mL, doses tested for induction werefrom 36.3 to 500 µg/mL in the first experiment, then from 36.3 to 228 µg/mL in the last two experiments. In the first experiment, a dose increase was noticed for the CD54 expression with the test item at any tested non-cytotoxic concentrations. No increase of expression for CD86 marker was observed. In the last two experiments, a dose-response relationship was noticed for CD54 marker with an increase of 2.27 to 11.06 fold of expression compared to the negative control. No increase of expression for CD86 marker was observed.

Based on these results, the test item “Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts” demonstrated an in vitro sensitizing potential with a Minimum Induction Threshold (MIT) of 43.0 µg/mL under the conditions in this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

From the DPRA a negative conclusion could be drawn notwithstanding partial co-elution with both the cysteine- and lysine-containing peptides took place. TheKeratinoSens^TMassay gave a clear negative result, while the h-CLAT gave a clear positive result in the form of CD54 induction. Based on the two negatives out of three results from these assays, the test substance “Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-hydroxyethyl), hydroxides, sodium salts” (CAS No. 70983-43-6) can be concluded to be negative for skin sensitization.

The substance does not have to be classified for skin sensitization according to Regulation EC No 1272/2008 and amendments.