1,2-Ethandiylbis(oxy-2,1-ethandiyl)-dimethansulfonat

Endpoint summary

Description of key information

Description of key information

• QSAR DEREK (in silico): Skin sensitation is plausible for alert 414 (Alkyl sulphate or sulphonate); Substance is predicted to be a skin sensitizer.


• DPRA (OECD 442C, in chemico): Mean peptide depletion: 12.30 %; Positive (low reactivity class).


• Keratinosens (OECD 442D, in vitro): Negative, non sensitizer; No cytotoxicity observed; Imax: 2.95 and 2.35 fold induction

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

17 February 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE: DEREK NEXUS

2. MODEL (incl. version number): DEREK NEXUS 6.1.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: CS(=O)(=O)OCCOCCOCCOS(=O)(=O)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: See the QMRF in the study document attached

5. APPLICABILITY DOMAIN
- Descriptor domain: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be conside red to be within the applicability domain. The applicability of potency predicitons may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compoint does not activate and alert or reasoning tule then Derek makes a negative prediction. The applicability of the negative prediction to the query compoinds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
- Structural fragment domain: The substructures of the substance fall within the structural fragment domain for alert 414.
- Mechanism domain: The structural fragments of the substance fall within the mechanistic domain of the molecules in the training set on which the alert is based.
- Metabolic domain: Not applicable, as the skin sensitization mechanism of the structural alert is not based on metabolism.

6. ADEQUACY OF THE RESULT
- Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments.
- Approach for regulatory interpretation of the model result: This result can be used within a weight-of-evidence approach to complete the endpoint skin sensitization.
- Outcome: Substance should be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.
- Conclusion: The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitization.

Qualifier:

according to guideline

Guideline:

other: Prediction on the potential for skin sensitisation with the model DEREK NEXUS

Version / remarks:

Version 6.1.1

Deviations:

no

Principles of method if other than guideline:

- Knowledge Base: Derek KB 2018 1.1
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

GLP compliance:

no

Specific details on test material used for the study:

SMILES: CS(=O)(=O)OCCOCCOCCOS(=O)(=O)C

Key result

Parameter:

other: Alert for skin sensitation

Remarks on result:

other: DEREK NEXUS triggered a skin sensitization alert 414 “Alkyl sulphate or sulphonate” for Triethylene glycol dimethanesulfonate based on the presence of two sulphonate substructures and predicted the substance to be sensitizing to the skin (plausible).

Interpretation of results:

other: Substance is predicted to be a skin sensitizer according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

Conclusions:

DEREK NEXUS Version 6.1.1. triggered a skin sensitization alert 414 “Alkyl sulphate or sulphonate” for Triethylene glycol dimethanesulfonate (CAS 80311-823) based on the presence of two sulphonate substructures and predicted the substance to be sensitizing to the skin (plausible).

Executive summary:

DEREK NEXUS version 6.1.1 triggered a skin sensitization alert 414 “Alkyl sulphate or sulphonate” for Triethylene glycol dimethanesulfonate based on the presence of two sulphonate substructures and predicted the substance to be sensitizing to the skin (plausible). The potential mechanism behind the alert is that the substance can directly alkylate nucleophilic centers in skin proteins.

DEREK NEXUS predicted an EC3 of 2.2% (GHS 1B) for Triethylene glycol dimethanesulfonate based on LLNA data from ten structurally related analogues (structural similarity of 18 - 46%). Because the query structure has two reactive sulphonates it may have an increased interaction with skin proteins compared to the structural analogues that contain one sulphonate group. Considering the EC3 was based on structurally related substances containing only one sulphonate or alkyl sulphate group it may be that the EC3 prediction of 2.2% (GHS 1B) is underestimated in DEREK.

In conclusion, Triethylene glycol dimethanesulfonate (CAS 80311-823) is predicted to be sensitizing to the skin. However, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH, but may be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2 -12 May 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM (INVITTOX) Protocol n°155: KeratinoSens™

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

Batch No.: 01110008
Storage: At room temperature (max 40 °C), protected from light

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PRINCIPLE OF THE KeratinoSens™ METHOD
The KeratinoSens™ method is an in vitro assay that quantifies the extent of luciferase gene induction following 48 hours incubation time of the KeratinoSens™ reporter cells with the test items. Luciferase gene induction is measured in the cell lysates by luminescence detection using a light producing luciferase substrate (Luciferase Reagent). Cytotoxicity and the relative luminescence intensity of luciferase substrate in the lysates are measured and luciferase induction compared to solvent/vehicle control is calculated.
KeratinoSens™ cells were derived from HaCaT human keratinocytes and transfected with selectable plasmids containing luciferase gene under the transcriptional control of the AKR1C2 ARE gene sequence, upstream of the SV40 promoter. AKR1C2 is known to be one of the genes up-regulated upon contacting skin sensitisers in dendritic cells. Therefore, this method is able to mimic the activation of the Keap1-Nrf2-ARE regulatory pathway and is relevant for the assessment of the skin sensitisation potential of test items. A prediction model is used, to support the discrimination between sensitisers and non-sensitisers.

PROCEDURE OF THE KeratinoSens™ METHOD
0. Solubility assessment of the test item & Preincubation of cells
1. Seeding of cells for testing - 24 h incubation
2. Preparation of the test item stock solution
3. Preparation of master plates
4. Exposure – 48 h incubation
5. Luciferase activation measurement
6. Cytotoxicity assessment

PREPARATION OF TEST SOLUTIONS
- Formulation of the test item: Possible solvents for the test item were dimethyl sulfoxide (DMSO) and sterile ultrapure water or exposure medium. Test item was first tried to be dissolved in DMSO to the final concentration of 200 mM. The DMSO solutions can be considered self-sterilising, so that no sterile filtration is needed. The test item could be properly dissolved in DMSO and a clear, transparent, homogenous solution was gained after vortexing. Ultrapure water was also tested as a possible solvent. After vortexing a clear, transparent, homogenous solution was obtained. Since DMSO is the preferred vehicle according to the test guideline, and the solution complied with all requirements, it was chosen as the solvent. The pre-experiments on solubility of the test item were not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the Final Report, but the raw data of these tests will be archived under the study code of present study.

- Test item Master Solutions: Based on the test item stock solutions made of DMSO, 2-fold serial dilutions in the first run and 1.2-fold serial dilutions in the second run (to be able to determine EC1.5 value more precisely) were made using the solvent to obtain twelve 100 × master concentrations of the test item creating a 100 × master plate. The 100 × master concentrations were further diluted 25-fold into exposure medium to obtain the 4 × master plate, by adding 10 µL of the 100 × master concentrations to 240 µL exposure medium.

- Positive control: The positive control used was Trans-Cinnamaldehyde for which a series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 µM.

- Negative control: The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the master and working solution concentrations in 6.3.1, so that the final negative (solvent) control concentration was 1 % DMSO in exposure medium on the treated plates. This DMSO concentration is known not to affect cell viability and corresponds to the same concentration of DMSO used in the tested chemical and in the positive control.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
After the 24-hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test item and control substances were added to each well in a way that an additional 4-fold dilution was achieved on the plate for the final concentrations to be established (50 µL of 4 × master solution to 150 µL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test item.

SEEDING AND INCUBATION
- KeratinoSens™ cell line
Name: KeratinoSens™ cell line
Description: immortalised adherent cell line derived from human keratinocytes (HaCaT) transfected with selectable plasmids
Supplier: Givaudan Schweiz AG
Lot Number: 20160415
Date of production: April 15, 2016

- KeratinoSens™ master culture
Subcultured MC4 master culture was used for the study.
ID of the cell line: KeratinoSens™ MC4 p5 20180308-20220420
Date of preparation: March 08, 2018
Date of thawing: April 20, 2022
Passage number at start: p9
Passage number at the end: p11
Storage: Vapor phase of liquid nitrogen

- Preparation of Media for KeratinoSens™ cells
Maintenance (culture) medium: DMEM supplemented with 9.0 (v/v) % fetal bovine serum (FBS) and ~ 500 µg/mL G418.
Thawing medium: DMEM containing 9.1 (v/v) % FBS without G418
Exposure medium: DMEM containing 1 (v/v) % FBS without G418

- Preparation of cells: Cells were subcultured upon reaching 80 - 90 % confluence and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium and distributed into 96-well plates (10 000 cells/well) homogenously. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wellsmicroplates at 37 ± 1 °C in the presence of 5 % CO2.

LUCIFERASE ACTIVITY MEASUREMENTS
After the 48-hour exposure time with the test item and control substances, cells were washed with DPBS (270 µL), and 1 × lysis buffer (20 µL) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 µL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.

CYTOTOXICITY
For the cell viability assay, medium was replaced after the 48-hour exposure time with MTT working solution (200 µL) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilised by the addition of isopropanol (50 µL). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.

DATA EVALUATION
The following parameters (endpoint values) are calculated in the KeratinoSens™ 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.

PREDICTION MODEL
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 tests, otherwise the KeratinoSens™ prediction is considered negative:
- the Imax is equal or higher than 1.5-fold and statistically significantly different as compared to the negative/solvent control (as determined by a two-tailed, unpaired Student’s T-test);
- the cellular viability is equal or higher than 70 % at the lowest concentration with induction of luciferase activity >= 1.5-fold;
- the EC1.5 value is less than 1000 µM (or < 200 µg/mL for test items with no defined molecular weight);
- there is an apparent overall dose-response for luciferase induction (or a biphasic response).

If in a given test, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, then the result of that test should be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 1000 µM (or <200 µg/mL for test items with no defined molecular weight) and which do not reach cytotoxicity (< 70 % viability) at the maximal tested concentration should also be considered as inconclusive.
In cases, if the test item induces the luciferase activity very close to the cytotoxic levels, it might be positive in some tests at non-cytotoxic levels and in other tests only at cytotoxic levels. Such test items shall be retested with a narrower dose-response analysis, using a lower dilution factor between wells, to determine if induction has occurred at cytotoxic levels or not.
With respect to chemicals tested at lower concentrations than the default ones, results fulfilling the criteria for positivity could still be used to support the identification of the test item as a skin sensitiser. In cases where a negative result is obtained in a test with a maximal concentration lower than 1000 µM and no cytotoxicity is reached, the result should be considered as inconclusive. If cytotoxicity (< 70 % viability) is reached at a maximal soluble test concentration lower than 1000 µM, criteria for negativity can still be applied.

The prediction model of the KeratinoSens™ assay requires multiple runs. For the assessment of whether the outcome of repeated runs yields a positive, negative or borderline final outcome in KeratinoSens™ the prediction model in Figure 2 shall be applied (adapted from the prediction model described in TG 442D to be used within the 2 out of three 3 defined approach (2o3 DA) to conclude on borderline cases). This prediction model introduces a third outcome (borderline) to be used within the 2o3 DA, based on the same decision cut-offs of the prediction model described in TG 442D. Thus, a negative in the original prediction model can only become negative or borderline, while a positive from the original prediction model can only become positive or borderline. The original threshold for a positive classification is 1.5-fold induction, and the statistically derived borderline range around this threshold is 1.35 – 1.67-fold.

Vehicle / solvent control:

DMSO

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at several concentrations in both tests. Although the luciferase activity induction in this first run was outside of the 2 – 8-fold induction range, there was a clear dose response relationship in the luciferase activity induction for the positive control, therefore it was accepted as valid.
The EC1.5 values of the positive control fell between 7 µM and 30 µM (17 µM and 24 µM for the first and second test).

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

2.95

Cell viability:

The test item induced no cytotoxicity (or viability below 70 %) compared to the solvent/vehicle control at any concentration in KeratinoSens™ cells.

Vehicle controls validity:

valid

Remarks:

Coefficient of variation (%) 7.9

Positive controls validity:

valid

Remarks:

Imax = 20.63

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

2.35

Cell viability:

The test item induced no cytotoxicity (or viability below 70 %) compared to the solvent/vehicle control at any concentration in KeratinoSens™ cells.

Vehicle controls validity:

valid

Remarks:

Coefficient of variation (%) 8.5

Positive controls validity:

valid

Remarks:

Imax = 3.66

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

For the test item, twelve doses ranging from 2000 µM to 0.98 µM were used in the first test.
In order to be able to determine the EC1.5 value more precisely, a narrower dilution factor was used in the second test and twelve doses ranging from 2000 µM to 269.2 µM were used.
The test item induced no cytotoxicity (a substance is considered cytotoxic if the viability is below 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, in none of the tests IC30 or IC50 values could be determined.
However, the induction values of the test item exceeded the 1.5-fold threshold at the higher tested concentrations in both tests:
The maximal fold induction (Imax) were 2.95 2.35-fold in the first and second test, respectively (these values are higher than the borderline threshold range) and the EC1.5 values were determined as 1071 µM for the first and 1121 µM for the second test. Moreover, a clear dose response could be observed in both tests. Nevertheless, both valid tests were concluded negative because the EC1.5 values were higher than 1000 µM in both cases.

Summary of the KeratinoSens™ results:

Number of tests	Significant induction above 1.5-fold (yes/no)	Viability ≥ 70 % at lowest concentration with ≥ 1.5-fold (yes/no)	EC1.5 < 1000 μM or 200 μg/ml (yes/no)	Showing clear dose response (yes/no)	KeratinoSens™ result obtained (positive/negative/ borderline/ inconclusive-repeat)
1	yes	yes	1071	yes	negative*
2	yes	yes	1121	yes	negative*
OVERALL CONCLUSION					negative

*The result of both tests is concluded negative, since the EC1.5 values are higher than 1000 μM.

Interpretation of results:

other: no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in k eratinocytes

Conclusions:

Based on these results and the KeratinoSens™ prediction model, the test item “Triethylene glycol dimethanesulfonate” (CAS 80322-82-3) is concluded negative for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Executive summary:

In the course of this study the skin sensitization potential of the test item “Triethylene glycol dimethanesulfonate” (CAS 80322-82-3) was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

In order to derive a prediction for the test item the results of two independent tests were used. Since the results of the two tests were concordant, a third was not needed in order to derive a conclusion.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.

For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in the first test. In order to be able to determine the EC1.5 value more precisely, a narrower dilution factor was used in the second test and twelve doses ranging from 2000 μM to 269.2 μM were used.

 

The test item induced no cytotoxicity (a substance is considered cytotoxic if the viability is below 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, in none of the tests IC30 or IC50 values could be determined.

 

However, the induction values of the test item exceeded the 1.5-fold threshold at the higher tested concentrations; EC1.5 values could be determined and clear dose responses were observed; both valid tests were concluded negative because the EC1.5 values and the overall
EC1.5 value were higher than 1000 μM in both cases.

 

Based on these results and the KeratinoSens™ prediction model, the test item “Triethylene glycol dimethanesulfonate” (CAS 80322-82-3) is concluded negative for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 - 22 April 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:

14 June 2021

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM (INVITTOX) Protocol 154: Direct Peptide Reactivity assay (DPRA) for skin sensitisat ion testing

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SANCO Guideline 3030/99

Version / remarks:

rev.4, July 11, 2000
Technical Material and Preparations: Guidance for generating and reporting
methods of analysis in support of pre- and postregistration data requirements for Annex II (part A,
Section 4) and Annex III (part A, Section 5) of Directive 91/414

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Batch No.: 01110008
Storage: At room temperature (max 40 °C), protected from light

Details on the study design:

PRINCIPLE OF THE DPRA METHOD
The reactivity of a test chemical and synthetic Cysteine or Lysine containing peptides was evaluated by combining the test chemical with a solution of the peptide and monitoring the remaining concentration of the peptide following 24 hours of interaction time at room temperature.The peptide was a custom material containing phenylalanine to aid the detection and either Cysteine (“C”) or Lysine (“K”) as the reactive centre.
Relative concentrations of the peptides following the 24 hour incubation were determined by high performance liquid chromatography with gradient elution and UV detection at 220 nm. Reaction samples, reference controls A, B and C, co-elution controls and positive controls are prepared and analysed in triplicates in batches of up to 26 chemicals or up to 30 hours run. The HPLC analysis time was within 30 hours.

STEPS OF THE DPRA METHOD DONE IN CHRONOLOGICAL ORDER
- Solubility assessment of test item
- Preparation of buffer solutions
- Pre-weighting of test item and positive control
- Pre-weighting of cysteine or lysine peptide for stock solution
- Test item and positive control solution preparation
- Peptide stock solution preparation
- Calibration standards preparation by serial dilution using dilution buffer
- Assembling of standards, reaction samples, positive controls, reference controls (A, B and C) and co-elution controls. For each set of control/sample replicates, the triplicate vials are prepared individually but from the same solutions.
- Preparation of HPLC system (column equilibration)
- HPLC analysis
- Data evaluation

PREPARATION OF TEST SOLUTIONS
- Formulation of the test item
The solubility of the test item was tested in a non-GLP preliminary solubility test evaluated at the concentration of 100 mM.
Acetonitrile was used first as a possible solvent and it dissolved the test item completely. A transparent and clear solution was formed immediately at 100 mM. The test item was dissolved also in ultrapure water completely, and also a transparent, clear solution was obtained.
The behavior of the formulation of the test item with acetonitrile was determined in the buffers of the test system (phosphate and acetate buffer) in a ratio corresponding to the reaction sample assembly. The compatibility of the formulation with the buffers of the test system was proven, no precipitate was observed in any cases and homogenous, clear solutions were obtained. Since acetonitrile is the first preferred vehicle in the order of solvents listed in the guideline and the formulation complied with all obligations of the test guideline, acetonitrile was chosen as the solvent.

- Synthetic petptides
* Cysteine peptide
Name: Cysteine peptide
Batch: 111016HS-MHeW0222-01
Storage: at -20°C or below
Purity: 93.98 %
Molecular weight: 750.88 g/mol
Sequence: Ac-RFAACAA-OH
Expiry date: September 01, 2022
Supplier: JPT

* Lysine peptide
Name: Lysine peptide
Batch: 020517HS-MHeW0222-01
Storage: at -20°C or below
Purity: 94.34 %
Molecular weight: 775.91 g/mol
Sequence: Ac-RFAAKAA-OH
Expiry date: September 01, 2022
Supplier: JPT

Assembly of reaction samples and controls
- 1:10 ratio cysteine peptide (0.5 mM peptide, 5 mM test item)
* 750 µL cysteine peptide stock solution (or phosphate buffer for the co-elution control)
* 200 µL acetonitrile
* 50 µL 100mM test item solution (or solvent for the reference controls A,B,C or 100 mM positive control solution for the positive control)
- 1:50 ratio lysine peptide (0.5 mM peptide, 25 mM test item)
* 750 µL lysine peptide stock solution (or ammonium acetate buffer for the co-elution control)
* 250 µL 100mM test item solution (or solvent for the reference controls A,B,C or 100 mM positive control solution for the positive control)

The autosampler vials were capped, vortexed to mix and placed to the HPLC autosampler tray for incubation at 25°C in the dark. HPLC analysis of the reaction samples was started 24±2 hours after the test item was added to the peptide solution. The samples were clear and homogenous after the incubation period. System equilibration was applied with the eluents of 50% phase A and 50% phase B for 2 hours and running the gradient twice before injecting the first sample. The column oven was 30°C, the measurements were performed at 25°C. The batches were consisted of 2 parts: one part with the A reference controls and the first sampling of the calibration standards. These samples were run before the 24±2 hours incubation time ends and right before the other part started. The other part contained the B and C reference controls, the positive controls, the reaction samples, the other sampling of the calibration standards and the co-elution controls. These samples were run right after the 24±2 hours incubation time ended. The total HPLC analysis time was less than 30 hours.

PREPARATION OF THE HPLC
HPLC system: SHIMADZU LC2030 (Prominence-i LC-2030C)
Serial number: L21445402951AE
Detector: 220 nm – D2 lamp
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 µm)
Serial number: USRY003976
Column temperature: 30°C
Sample temperature: 25°C
Injection volume: 7µL
System equilibration: 50% phase A and 50% phase B for 2 hours at 30°C and running the gradient twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow
Mobile phases for HPLC:
Mobile Phase A – 0.1 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile

DATA EVALUATION
- Percent peptide depletion
The concentration of the peptide was determined in each sample and positive control, from absorbance at 220 nm measuring the peak area of the appropriate peaks and calculating the concentration of the peptide using the linear calibration curves derived from the calibration standards.
The percent peptide depletion is determined in each reaction sample and positive control measuring the quotient of the peak area and the mean respective reference control C peak area, according to the formula described below:
peptide percent depletion = [1- { (peak area of the reaction sample or pc) / (mean peak area of reference controls C) } ] × 100

- Presence of precipitate
If precipitation occurs it is recorded and caution is used in interpreting data. Samples can be centrifuged to settle and remove the precipitate to avoid clogging the HPLC. Centrifugation at low speed (max. 400 x g) is recorded as well.

- Co-elution
In cases where a test chemical co-elutes with the lysine peptide, the Cysteine 1:10-only prediction model can be used. In cases where the test chemical co-elutes with the cysteine peptide and the peptide peak cannot be integrated, a determination of reactivity cannot be made based on the Percent Depletion data from the lysine reaction alone, and the data is reported as “inconclusive”. If the peak for the cysteine peak can be integrated, the instructions below are followed to determine an estimated Percent Depletion.

- Negative depletion values
If the Percent Peptide Depletion is < - 10.0%, it is considered that this may be a situation of co-elution, inaccurate peptide addition to the reaction mixture or just baseline “noise.” If this happens, the co-elution controls are carefully analyzed. If the peptide peak appears at the proper retention time and has the appropriate peak shape, the peak can be integrated. The calculated percent peptide depletion is reported as an “estimate“. If this was only an issue for lysine, use the “cysteine-only” prediction model.
If the peak does not have the proper shape due to complete overlap in retention time of the test chemical and peptide and cannot be integrated, calculation of Percent Peptide Depletion is not possible. If this is an issue for lysine, use the “cysteine-only” model. If this is an issue for cysteine or both cysteine and lysine, the data is reported as “inconclusive”.

- Co-elution Controls
If a chemical (Co-elution Control) absorbs at 220 nm and has a similar retention time as a peptide (Reference Control) and the peaks are overlapping, then co-elution of the test chemical with the peptide is reported.
In order to assure that baseline noise is not being called interference it is checked, if the “interfering” chemical peak has a peak area that is >10% of the mean peptide peak area in the appropriate Reference Control. If co-elution occurs, proper integration and calculation of Percent Peptide Depletion is not possible. The data is recorded as “interference” for that peptide.

- Co-elution with reactivity and estimated depletion values
In some instances, a test chemical may have an overlapping retention time with either of the peptides and still be reactive with that peptide. This can make the peak area of the peptide appear to be larger than it really is, therefore the calculated percent depletion may be underestimated. If this is the case and the overlap in retention time between the test chemical and peptide is incomplete, percent depletion can still be calculated with a notation of “coelution – percent depletion estimated”.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

The acceptance criteria were met for the positive control with a cysteine peptide depletion value
of 67.76 % ± 2.42 % and with a lysine peptide depletion value of 50.22 % ± 4.29 %.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Remarks:

three replicates

Value:

21.92 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

2.68 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

low reactivity [in chemico]

Other effects / acceptance of results:

REJECTED RUNS AND FAILURE TO MEET ACCEPTANCE CRITERIA
No runs were rejected.

CO-ELUTION
No co-elution to any content was observed with either the cysteine or lysine peptide.

SYSTEM SUITABILITY
Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior analysis. The mean peptide concentration of A reference control sample replicates was 0.52 mM for the cysteine and 0.50 mM for the lysine peptides.

CALIBRATION CURVE
Six concentration levels were used: 0.0167, 0.0334, 0.0668, 0.1335, 0.2670 and 0.5340 mM. One such series were prepared from both peptides and analysed with three replicate injections from each solution.
Peak area versus the concentration of both peptides showed good linearity in the range of 0.0167 – 0.5340 mM. The calibration graph was calculated applying linear regression. The coefficients of the determination (r2) were 0.9997 thus the calibration models fulfilled the linearity requirements.

ANALYSIS SEQUENCES
Reference control B replicates were included in the sequence to verify the stability of the peptide over time and reference control C replicates were used to verify that the solvent of the test item did not impact the percent peptide depletion.
The CV % for the nine reference control B and C replicates in acetonitrile (acn) was 3.1 % and 0.6 % for cysteine and lysine peptides respectively. The mean peptide concentration of B and C reference control sample replicates was 0.52 mM for the cysteine and 0.50 mM for the lysine peptides.

CYSTEINE AND LYSINE DEPLETION AND MEAN PEPTIDE DEPLETION OF THE TEST ITEM AND POSITIVE CONTROL
The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 67.76 % ± 2.42 % and with a lysine peptide depletion value of 50.22 % ± 4.29 %.
The acceptance criteria were met for the mean peptide concentrations of the reference control C with 0.52 mM for the cysteine and 0.50 mM for the lysine peptides, respectively.
The mean cysteine peptide depletion value was 21.92 % ± 10.04 while the lysine peptide depletion value of the test item was 2.68 % ± 0.64 %.

ASSIGNING THE TEST CHEMICAL TO A REACTIVITY CLASS AND CATEGORY
The average percent peptide depletion was calculated for the test item. No co-elution was observed with the peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The overall mean peptide depletion of the test item was 12.30 %, which exceeds the 6.38 % threshold of the applicable prediction models and thus was classified as positive (low reactivity). Because the mean peptide depletion of 12.30 % also exceeds the range of 3% to 10%, a second run is not required.

Interpretation of results:

other: positive (low reactivity)

Conclusions:

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “Triethylene glycol dimethanesulfonate” (CAS 817-828-0) was concluded to be positive (low reactivity) under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

Executive summary:

In the course of this study the skin sensitization potential of the test item “Triethylene glycol dimethanesulfonate” (CAS 817-828-0) was studied using the Direct Peptide Reactivity Assay (DPRA).

For the test item and positive control substance, in order to derive a prediction, two independent tests were evaluated, one with cysteine and one with lysine peptides.

Peptide depletion resulting from the positive control cinnamaldehyde was within the expected percentage range both with cysteine and lysine peptides. Peptide depletion resulted from the positive control cinnamaldehyde was 67.76 % ± 2.42 % with cysteine peptide and 50.22 % ± 4.29 % with the lysine peptide.

The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for the cystein run (0.50 mM) and lysine run (0.52 mM) and the CV % values for the nine reference controls B and C in acetonitrile were 3.1 % and 0.6 % for the cysteine and lysine runs, respectively. For each peptide, all validity criteria were met, confirming the validity of the study.

The mean cysteine peptide depletion value was 21.92 % ± 10.04 % while the lysine peptide depletion value of the test item was 2.68 % ± 0.64 %.

The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The overall mean peptide depletion of the test item was 12.30 %.

 

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “Triethylene glycol dimethanesulfonate” (CAS 817-828-0) was concluded to be positive (low reactivity) under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

DEREK NEXUS version 6.1.1 did yield a skin sensitization alert for the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) based on the presence of two sulphonate substructures and predicted the test item to be sensitizing to the skin (plausible). DEREK NEXUS predicted an EC3 of 2.2 % (moderate sensitizer, GHS 1B) for the test item based on LLNA data from ten structurally related analogues which however contained only one (instead of two) reactive sulphonate groups. Therefore, the EC3 prediction may be underestimated by DEREK NEXUS.

A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in acetonitrile at 100 mM. There was no evidence of co-elution of the test item with either Cysteine or Lysine peptide. Peptide depletion was calculated as 21.92 % +/- 10.04 % and 2.68 % +/- 0.64 % for Cysteine and Lysine peptides, respectively, resulting in a mean peptide depletion of 12.30 %. This value places the test item in the ‘Low Reactivity’ class, resulting in a DPRA prediction of "sensitiser" when using the Cysteine 1:10/Lysine 1:50 prediction model.

A valid KeratinoSens^TM assay was performed according to OECD 442D and GLP principles. To derive a prediction for the test item the results of two independent tests were used. The test item was suspended in dimethyl sulfoxide (DMSO) at 200 mM. In the first test, twelve doses ranging from 0.98 – 2000 µM of the test item were used (2-fold dilution series). To determine the EC1.5 value more precisely, a narrower dilution factor was used in the second test, and twelve doses ranging from 269.2 – 2000 μM of the test item were used (1.2-fold dilution series). No precipitation was observed at any point during the two tests. The test item showed no toxicity (no IC30 and IC50 value). The luciferase activity induction exceeded the threshold of 1.5-fold compared to the respective negative control in both tests (in test 1 at 2000 μM, in test 2 in the concentration range of 1157.4 – 2000 μM). The maximum luciferase activity induction (Imax) was higher in both tests than the borderline threshold range with 2.95-fold and 2.35-fold in test 1 and 2, respectively. A clear dose response could be observed for both tests. Both tests were concluded negative because the EC1.5 value was higher than 1000 μM. Therefore, the test item is concluded negative for skin sensitization potential under the experimental conditions of KeratinoSens^TM assay method.

Justification for classification or non-classification

DEREK NEXUS predicts the test item as a moderate sensitizer (GHS 1B) with at least equivocal probability. The DEREK prediction was based on LLNA data of structurally related substances classified as sensitizers. However, due to an additional reactive sulphonite group in the test item when compared to the structurally related substances, the DEREK prediction may be underestimated.

Furthermore, based on the results of OECD 442C, the test item is expected to bind to skin proteins (triggering the first key event of the adverse outcome pathway), supporting the DEREK prediction of the test item being a weak-to-moderate skin sensitiser.

In the OECD 442D (assessing the second key event), a significant induction above 1.5-fold at a viability > 70 % as well as a clear dose response to the test item was observed. However, the test was concluded to be negative for skin sensitisation due to the EC1.5 values which exceeded the threshold of 1000 µM.

Therefore, equivocal results were achieved in the in silico/in chemico and the in vitro assays. However, based on the score-based “Integrated Testing Strategy (ITS) Defined Approach (DA)” prediction model described in OECD 497, the test item can nevertheless be considered as a moderate-to-weak skin sensitiser based on the following reasoning:             
Both the positive DEREK prediction as well as the mean peptide depletion of 12.30 % observed in the DPRA result in scores of 1 (overall score 2). According to OECD Guideline 497 paragraph 109, a DA prediction based on one in chemico/in vitro and one in silico prediction with a total score of 2 (UN GHS 1B) is conclusive as the outcome of an additional in vitro method would not result in a different DA prediction.

In conclusion, the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) is considered to be a moderate/weak skin sensitizer (UN GHS Cat. 1B) according to Regulation 1272/2008 and amendments.