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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No in vitro mutagenicity response has been observed in following test systems:

- A bacterial reverse mutation test ( OECD 471)was conducted in S. typhimurium strains TA 1535, TA 1537, TA 98 , TA 100 and Ecoli WP2 with and without metabolic activation to assess the potential mutagenic effect of Reaction  mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate (Charles River 2022 ). The test item  was not toxic towards the tester strains, therefore 5000 µg/plate was chosen as the top dose level in the mutation tests. No substantial increases in the revertant colony numbers of any of the 5 strains were observed following treatment with Reaction  mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix).

- An in vitro Cytogenetic Assay measuring chromosomal Aberration test (OECD 473) was conducted with Reaction  mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate  in human lymphocytes with and without metabolic activation up to concentrations of 2000 µg mL (Charles River 2022). No biological relevant effect of the test material on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.  Therefore it can be concluded that the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. Hence, the test item  was considered as non-mutagenic in in vitro chromosomal aberration assay using human lymphocytes.

- An in vitro mammalian cell gene mutation assay (OECD 490) was conducted  with Reaction  mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate in the L5178Y TK+/- mouse lymphoma cell line with and without metabolic fraction at concentrations of 25,50,100,250,500,1000,1500,2000 µg/mL (Charles River 2022). In the absence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency in the first experiment. In the presence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency.In conclusion, Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions .

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

L5178Y TK+/- mouse lymphoma cell line with and without S9

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Specific details
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 %

Target gene:

Thymidine kinase (TK) locus in L5178 Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) or RPMI 1640 Hepes buffered medium (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.
Exposure medium
Cells will be exposed to the test material in basic medium supplemented with 5% to 10% (v/v) heat-inactivated horse serum.
Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT) (Sigma).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight)

Test concentrations with justification for top dose:

Dose range finder.

In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test material concentration range of 125 to 2000 µg/mL(0,125,250,500,1000,2000 µ/L) in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The highest concentration which did not precipitate in the exposure medium was 2000 μg/mL. The pH and osmolarity at a concentration of 2000 μg/mL were 7.41 and 0.320 Osm/kg respectively (compared to 7.42 and 0.296 Osm/kg in the solvent control).Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 2000 μg/mL compared to the solvent control.

Mutation experiment

Eight doses (25,50,100,250,500,1000,1500,2000 µg/ml)of the test material were tested in the mutation assay. The test material was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods. The top concentration was 2000 µg/mL.

Vehicle / solvent:

The vehicle of the test material was Milli-Q water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

The negative control was Milli-Q water, the vehicle of the test material.

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

Experimental Design
Cleansing

Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in growth medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on growth medium containing hypoxanthine and thymidine only. After this period cells were returned to growth medium for at least 1 day before starting the experiment.

Dose-range Finding Test

In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells
(1.25 x 105 cells/mL for 24 hour treatment) with a number of test material concentrations increasing by approximately half log steps. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test material was tested in the absence and presence of S9-mix.
The highest tested concentration was 2000 µg/mL exposure medium.
For the 3 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium.
For the 24 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium. The cells in the final suspension were counted with the coulter particle counter.
The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity. After 24 hour of subculturing, the cells were counted and subcultured again for another
24 hours, after that the cells were counted. The surviving cells of the 24 hour treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than
1.25 x 105 cells/mL were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.

Mutagenicity Test

Eight doses of the test material were tested in the mutation assay. The test material was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with
3 and 24 hour treatment periods.
The top concentration was 2000 µg/mL.
Initially, a first mutation experiment was performed with a 3 hour treatment period in the presence of S9-mix. However, the solvent and positive control acceptability criteria were not met, therefore the first experiment with a 3 hour treatment period in the presence of S9-mix was rejected, and no data were reported. A repeat experiment for the 3 hour treatment in the presence of S9-mix was performed.
Treatment of the Cells
Per culture 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to the test material in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test material in exposure medium for 24 hours in the absence of S9-mix.
For the 3 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium.
For the 24 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium. The cells in the final suspension were counted with the coulter particle counter.

Expression Period

For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test material the cells were plated for determination of the cloning efficiency (CEday2) and the mutant frequency (MF).

Determination of the Mutant Frequency

For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a
96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in
non-selective medium.
For determination of the mutant frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
Analysis of Results
Determination of the Mutant Colonies
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.
Calculation of the Survival or Viability

Dose-range finding test:

The suspension growth (SG) for the 3 hour treatment=
SG = Suspension growth = [Day 1 cell count/1.6 x 105] x [Day 2 cell count/1.25 x 105]

The suspension growth (SG) for the 24 hour treatment=
SG = Suspension growth = [Day 0 cell count/1.25 x 105] x [Day 1 cell count/1.25 x 105]

Mutagenicity tests:
The suspension growth (SG) for the 3 hour treatment=
[Day 1 cell count/1.6 x 105] x [Day 2 cell count/1.25 x 105]

The suspension growth (SG) for the 24 hour treatment=
[Day 0 cell count/1.25 x 105] x [Day 1 cell count/1.25 x 105] x [Day 2 cell count/1.25 x 105]

Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100

The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells

The cloning efficiency (CE) was then calculated as follows:
CE = -ln P(0)/number of cells plated per well

The relative cloning efficiency (RCE) at the time of mutant selection =
CE (test) / CE (controls) x 100

The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture:
RTG = RSG x RCE/100
Calculation of the Mutant Frequency
The mutant frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutant frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 106
Small and large colony mutation frequencies were calculated in an identical manner.

ACCEPTABILITY CRITERIA

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutant frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
The positive control should demonstrate an absolute increase in the total mutant frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least

Evaluation criteria:

ACCEPTABILITY CRITERIA

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutant frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
The positive control should demonstrate an absolute increase in the total mutant frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10-6).

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test material is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test material is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutant frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

24 h exposure

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

3h exposure

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):

In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test material concentration range of 125 to 2000 µg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.The highest concentration which did not precipitate in the exposure medium was 2000 μg/mL. The pH and osmolarity at a concentration of 2000 μg/mL were 7.41 and 0.320 Osm/kg respectively (compared to 7.42 and 0.296 Osm/kg in the solvent control).Table 1 shows the cell counts of the cultures from the 3 hours of treatment with various concentrations of the test material after 24 and 48 hours of subculture, the calculated suspension growth and the relative suspension growth. Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 2000 μg/mL compared to the solvent control.Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of the test material and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth.
No toxicity in the relative suspension growth was observed up to test material concentrations of 2000 μg/mL compared to the solvent control

STUDY RESULTS
- Concurrent vehicle negative and positive control data

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (see Table 6). It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Study results and interpretation.
Table 3 and Table 4 show the percentages of cell survival and the mutation frequencies for various concentrations of the test material. Individual colony counts of cloning and selective plates and cell counts during subculturing are listed in Table 7 to Table 11 of Appendix 4
Based on the results of the dose-range finding test, the test material was tested in two mutation assays. The first experiment was performed in the absence and presence of S9-mix with a 3 hour treatment period. The second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. The following dose-range was selected for the mutagenicity tests in the absence and presence of S9-mix: 25, 50, 100, 250, 500, 1000, 1500 and 2000 μg/mL exposure medium.

Evaluation of toxicity
No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.

Evaluation of the mutagenicity
No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test material either in the absence or in the presence of S9-mix in both experiments. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Conclusions:

In conclusion, Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate is not mutagenic in the TK mutation test system under the experimental conditions described in this report

Executive summary:

The objective of this study was to evaluate the mutagenic potential of Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.  

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

Batch 202108300033 of the test material was a clear light-yellow liquid. A correction factor of 2.971 was used to correct for the purity. The vehicle of the test material was Milli-Q water.

The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 90%-110%). The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%). In the vehicle, no test material was detected.

In the first experiment, the test material was tested up to concentrations of 2000 µg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, the test material was again tested up to concentrations of 2000 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix.

The mutant frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical concurrent solvent control database, except in the second experiment in which the cloning efficiency of one of the solvent control cultures was not within the range of the acceptability criteria. Since the cloning efficiency was just above the higher limit of the acceptability criteria range and the cloning efficiency of the other solvent control culture was within the acceptability criteria range, this deviation in the mutant frequency had no effect on the validity of the results of the second mutation experiment.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency.

In conclusion, Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted in 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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 %

Species / strain / cell type:

lymphocytes: cultured peripheral Human Lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
Cultured peripheral human lymphocytes were used as test system.
Blood was collected from healthy adult, non-smoking volunteers (approximately
18 to 35 years of age).

- Suitability of cells: Peripheral human lymphocytes are recommended in international guidelines (OECD).
- Normal cell cycle time (negative control):
The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2021) are presented below:
Dose-range finding study: age 30, AGT = 12.9 h
First cytogenetic assay: age 28, AGT = 12.5 h
Second cytogenetic assay: age 30, AGT = 12.7 h



For lymphocytes:
- Sex, age and number of blood donors: Dose-range finding study: age 30, AGT = 12.9 h
First cytogenetic assay: age 28, AGT = 12.5 h
Second cytogenetic assay: age 30, AGT = 12.7 h

- Whether whole blood or separated lymphocytes were used:
Whole blood
- Whether blood from different donors were pooled or not:
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
-supplier: Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen,
- source of S9
prepared from male Sprague Dawley rats livers that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies).
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL
S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was

- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

Dose range finder.
In the dose-range finding test blood cultures were treated with 63, 125, 250, 500, 1000 and 2000 µg test material/mL culture medium with and without S9-mix.

Justification for top dose.
A concentration of 2000 µg/mL showed no precipitation in the culture medium. Therefore, this concentration was used as the highest concentration of the test material.
The pH and osmolarity of a concentration of 2000 µg/mL were 7.86 and 308 mOsm/kg respectively (compared to 7.89 and 280 mOsm/kg in the solvent control).

First cytogenetic assay:
Based on the results of the dose-range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix : 500, 1000, and 2000 µg/mL culture medium
(3 h exposure time, 24 h fixation time).

Second cytogenetic assay:
To obtain more information about the possible clastogenicity of the test material, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to the test material in the absence of S9-mix for 24 hours. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 10, 500, 1000, 1500, and 2000 µg/mL culture medium
(24 h exposure time, 24 h fixation time).

Vehicle / solvent:

. The vehicle of the test material was Milli-Q water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive cont

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration :Duplicate
- Number of independent experiments. two main assays were performed

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium.
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 and 24 h
- Harvest time after the end of treatment (sampling/recovery times):
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. Colchicine (0.5 µg/mL medium) (Acros Organics, Geel, Belgium) addedd for the last 3 h .
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for
10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 300 (150 per replicate culture)
- Determination of polyploidy: yes
- Determination of endoreplication: yes
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analyzable concentrations were used for scoring of the cytogenetic assay. The highest concentration analyzed was the recommended 2000 µg/mL.

Rationale for test conditions:

In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose-range finding test. the test material was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test material for 3 h and 24 h in the absence of S9-mix or for 3 h in the presence of
S9-mix. A negative control was included at each exposure time.
The highest tested concentration was the recommended 2000 µg/mL.
After 3 h exposure to the test material in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h fixation time).
Cytotoxicity of the test material in the lymphocyte cultures was determined using the mitotic index.
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 2000 µg/mL.

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:

a)At least 300 well spread metaphases were scored per concentration (Table 3, Table 4, Table 6)
b)The concurrent negative control data are considered acceptable as they are within the 95% control limits of the distribution of the historical negative control database (Table 7).
c)The concurrent positive controls induced responses that are compatible with those generated in the historical positive control database (Table 8).
d)The positive control material induced a statistically significant increase in the number of cells with chromosome aberrations, analyzed by the Fisher’s exact test (one-sided, p < 0.05) (Appendix 4).
e)The high dose was the highest dose required by the OECD 473 guideline (2000 µg/mL)
f) The lowest doses examined showed little or no toxicity.

Statistics:

Graphpad Prism version 8.4 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test material is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test material is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes: cultured peripherical human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

All acceptability criteria are met.
At least 300 well spread metaphases were scored per concentration (Table 3, Table 4, Table 6)
The concurrent negative control data are considered acceptable as they are within the 95% control limits of the distribution of the historical negative control database (Table 7).
The concurrent positive controls induced responses that are compatible with those generated in the historical positive control database (Table 8).
The positive control material induced a statistically significant increase in the number of cells with chromosome aberrations, analyzed by the Fisher’s exact test (one-sided, p < 0.05) (Appendix 4).
The high dose was the highest dose required by the OECD 473 guideline (2000 µg/mL)
The lowest doses examined showed little or no toxicity.

Table 1 :Mitotic Index of Human Lymphocyte Cultures Treated with Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate in the Dose-Range Finding Test

	Absolute number of metaphases	Number of cells scored	Percentage of control
Without metabolic activation
3 h exposure ,24 h fixation
Control (water)	126	1000	100
663	123	1000	98
125	124	1000	98
250	108	1000	86
500	99	1000	79
1000	90	1000	71
2000	76	1000	60

24 h exposure time, 204 h fixation time

Conclusions:

In conclusion, this test is valid and Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The possible clastogenicity of the test material was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

Batch 202108300033 of the test material was a clear light-yellow liquid. A correction factor of 2.971 was used to correct for the purity. The vehicle of the test material was Milli-Q water.

The concentrations analyzed in the dose formulation samples were in agreement with target concentrations. In the vehicle, no test material was detected.  The dose formulation samples were homogeneous and found to be stable during storage at room temperature protected from light for at least 4 hours.

In the first cytogenetic assay, the test material was tested up to 2000 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix. This is the highest dose level recommended in the guideline for testing.

In the second cytogenetic assay, the test material was tested up to 2000 µg/mL for a 24 h continuous exposure time with a 24 h fixation time in the absence of S9-mix.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test material did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No biological relevant effect of the test material on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.  Therefore it can be concluded that the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. 

In conclusion, this test is valid and Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames study OECD 471

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

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 %

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

- source of S9
- method of preparation of S9 mix :
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per
10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of
S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the
S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment

- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of the test material used in the subsequent mutation assays was 5000 µg/plate.

Vehicle / solvent:

The negative control was Milli-Q water, the vehicle of the test material.

Negative solvent / vehicle controls:

yes

Remarks:

Mili -Q water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

ethylmethanesulphonate

methylmethanesulfonate

other: 2-aminoanthracene (2AA°

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable
- Test substance added in medium; in agar plate incorporation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: not applicable
- Exposure duration/duration of treatment: 48 h
- Harvest time after the end of treatment (sampling/recovery times): immediately after exposure

Evaluation criteria:

A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Accuracy
In the vehicle, no test material was detected.
The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
Homogeneity
The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).
Stability
Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature protected from light conditions for at least 4 hours.
Formulation analysis report is presented in Appendix 5.

Conclusions:

All bacterial strains showed negative responses over the entire dose-range, i.e. no dose-related increase in the number of revertants in two experiments.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.In conclusion, based on the results of this study it is concluded that Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 202108300033 of the test material was a clear light-yellow liquid. A correction factor of 2.971 was used to correct for the purity. The vehicle of the test material was Milli-Q water.

The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%). Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature protected from light conditions for at least 4 hours. In the vehicle, no test material was detected.

In the dose-range finding study(table 1), the test material was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP₂uvrA in the absence and presence of 5% (v/v) S9-mix.                                                  In the first mutation experiment(table 2), the test material was again tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98 in the absence and presence of 5% (v/v) S9-mix.                                                  In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 492 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA.(table 3)                                                In all three experiments the test material did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The test material did not induce a dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in the tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that Reaction mass of disodium 2,2’-oxydiethanesulfonate and sodium ethenesulfonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008),  Reaction  mass of disodium 2,2 oxydiethanesulfonate and sodium ethenesulfonate does not have to be classified and has no obligatory labelling requirement for genetic toxicity.