trisodium 2-(2-sulfoethoxy)ethane-1-sulfonate ethenesulfonate

Endpoint summary

Description of key information

An integrated  testing  strategy, using a battery  of  three studies addressing the different key  events of skin sensitisation is usesd. The following studeis were conduxcted.

1)In chemico Direct  peptite reactivity assay ( DPRA) . result: negative

2) In vitro KeratinoSens. result:negative

3) Cell  line activation test for  skin sensitization  (Usens): result negative.

It was concluded that the test item Reaction  mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate was  not considered to be a skin sensitizer

 

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:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

U937 cell line activation test (U-SENS™)

Specific details on test material used for the study:

Reaction mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate
Batch lot number:202108300033
Dry weight content : 33.65 %

Vehicle / solvent control:

cell culture medium

Negative control:

DL-Lactic acid

Positive control:

other: 2,4,6 Trinitrobenzenesulfonic acid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Cell viability:

The test item showed no cytotoxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: • No biologically relevant increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. No EC150 could be calculated and is considered to be higher than 200 µg/mL.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Cell viability:

The test item showed no cytotoxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No EC150 could be calculated and is considered to be higher than 200 µg/mL.

Outcome of the prediction model:

negative [in vitro/in chemico]

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate is classified as negative (no biologically relevant increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Executive summary:

The objective of this study is to evaluate the ability of the test item to increase the expression levels of CD86 cell surface marker in the U937 cell line activation Test (U-Sens™) assay.

The test item was evaluated for the ability to increase the expression levels of CD86 cell surface marker. An overview of the viability and CD86 cell surface marker activity is summarized in Table 1. The results of the positive, negative and vehicle controls are summarized in Table 2. An overview of EC₁₅₀ and CV₇₀ values is given in Table 3. The individual raw data are presented in Table 4 and Table 5.

Two independent experiments were performed. The cell viability before incubation with the test item was > 90% (98% and 97% in experiment 1 and 2, respectively). The cells were in these experiments incubated with the test item in a concentration range of 1.0 – 200 µg/mL. The increase of CD86 cell surface marker expression was assessed by measuring the amount fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide.

Experiment 1

• No precipitation was observed at the end of the incubation period in the 96-well plates.


• The test item showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV₇₀ values could be calculated and is considered to be higher than 200 µg/mL.


• No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. No EC₁₅₀ could be calculated and is considered to be higher than 200 µg/mL.


• The test item showed no colour interference.


• The positive control (TNBS) showed a S.I. ≥ 1033% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a  I. ≤ 88% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Experiment 2

• No precipitation was observed at the end of the incubation period in the 96-well plates.


• The test item showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV₇₀ values could be calculated and is considered to be higher than 200 µg/mL.


• No biologically relevant increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. No EC₁₅₀ could be calculated and is considered to be higher than 200 µg/mL. At the dose level of 100µg/mL, the test item showed a CD86 induction of 153%. However, as this is only just above the threshold of 150%, and only occurred at this dose level and not at the highest dose level, this minor increase is not considered to biologically relevant.


• The test item showed no colour interference.


• The positive control (TNBS) showed a S.I. ≥ 634% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a I. ≤ 148% in all wells and was non-cytotoxic at all concentrations (cell viability            ≥ 70%).

 

 

Both tests passed the acceptance criteria:

• At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (99% in experiment 1 and 2).


• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and
  ≤ 25% in both experiments.


• At least two out of three IgG1 values of untreated U937 cells fell within the range of
  ≥ 0.6% and < 1.5% in both experiments.


• No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.

In both experiments the positive and negative control were considered valid, and the positive control fell within the historical control data. Overall, it is concluded that the test conditions were adequate and that the test system functioned properly.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

amino acid derivative reactivity assay (ADRA)

Specific details on test material used for the study:

Reaction mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate
Batch lot number:202108300033
Dry weight content : 33.65 %, correction factor is 2.971. The test item is a multi-constituent
Composition.
Constituent1:Sodium ethylene sulphonate cas 3039-83-6: 75.01 %
Constituent 2 : Isethionate bisether, disodium salt cas 63440-92-6: 16.52%
Impurity 1:Isethionate sodium cas 1562-00-1:4.12%
Impurity 2:Sodium ethionate, disodium salt cas1562-03-4:0.62 %
Impurity 3:Sodium sulfate cas 7757-82-6 :2.76 %
Impurity 4:Sodium ethandisulfonate disodium salt cas 5325-43-9:0.64 %

Details on the study design:

1)Test systhem

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.
Recommended test system in the international OECD guideline for DPRA studies.
JPT Peptide Technologies GmbH, Berlin, Germany.
batch cysteine 111016HS-MHeW0721
batch lysine :020517HS-MheW0721

The peptides were stored in the freezer set to maintain -20°C for a maximum of 6 months.

2)Vehicle/ Pos Control
vehicle used: MQ water
Positive control :cinnamaldehyde

Vehicle / solvent:

water

Positive control:

cinnamic aldehyde

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Mean cysteine and lysine depletion

Value:

1.5 %

At concentration:

100 mM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cysteine 1:10 and Lysine 1:50 prediction model.

Other effects / acceptance of results:

The coefficient of determination (r2) of the SPCL standard calibration curve was 0.9992. As the r2 was >0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.496 ± 0.005 mM, the mean peptide concentration of Reference Controls C was 0.504 ± 0.004 mM and the mean peptide concentration of Reference Controls CMQ was 0.496 ± 0.007 mM. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) 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 1.1%. 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 32.74. The mean A220/A258 ratio ± 10% range was 29.47-36.01. 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 64.7% ± 0.7%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Conclusions:

The mean of the SPCC and SPCL depletion was 1.5% and as a result the test item 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.

Executive summary:

In conclusion, this DPRA test is valid. Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate 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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

Reaction mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate
Batch lot number:202108300033
Dry weight content : 33.65 %

Vehicle / solvent control:

DMSO

Positive control:

other: Ethylene dimethacrylate glycol

Positive control results:

• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (72 µM and 67 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.19-fold and 2.84-fold in experiment 1 and 2, respectively).

Key result

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.2 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.18 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (72 µM and 67 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.19-fold and 2.84-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.5% and 5.6% 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 no toxicity (no IC30 and IC50 value) and 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 1.18-fold and 1.20-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 400 µg/mL.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no toxicity (no IC30 and IC50 value) and 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 1.18-fold and 1.20-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 400 µg/mL.

Executive summary:

The objective of this study was to evaluate the ability of Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensÔ assay.

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

Batch 202108300033 of the test item was a clear light-yellow liquid with a purity of 33.65%.  A correction factor of 2.971 was used to correct for the purity (33.65%). The test item was dissolved in dimethyl sulfoxide (DMSO) at 40 mg/mL. 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 the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both tests passed the acceptance criteria:

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


• The EC₅ of the positive control was within two standard deviations of the historical mean (72 µM and 67 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.19-fold and 2.84-fold in experiment 1 and 2, respectively).


• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.5% and 5.6% 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 no toxicity (no IC₃₀ and IC₅₀ value) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.18-fold and 1.20-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 400 µg/mL.

In conclusion, Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The results available from the three in vitro tests of the sensitisation AOP test battery reveal three negative results (USENS OECD 442E,  KeratinoSens OECD 442D, and DPRA 442C). Based on these data, the test item does not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.