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

Genetic toxicity: in vitro

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Sodium ethylenesulphonate

EC Number:

221-242-5

EC Name:

Sodium ethylenesulphonate

Cas Number:

3039-83-6

Molecular formula:

C2H4O3S.Na

IUPAC Name:

sodium ethenesulfonate

Test material form:

liquid

Details on test material:

25% solution in water

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 %

Method

Target gene:

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

Species / strain

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.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Controls 2

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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.

Applicant's summary and conclusion

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.