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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

Cas Number:

80322-82-3

Molecular formula:

C8H18O8S2

IUPAC Name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

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

IUPAC Name:

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

Test material form:

liquid

Details on test material:

Appearance: Dark yellow liquid
Batch No.: 01110008
Storage: -5 – 40 °C, container tightly closed, protected from light

Specific details on test material used for the study:

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

In vitro test system

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]

Results and discussion

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).

In vitro / in chemico

Results

Results 1

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

Results 2

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).