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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-11 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997, corrected 26th June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

November 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

Cas Number:

80322-82-3

Molecular formula:

C8H18O8S2

IUPAC Name:

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

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

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

IUPAC Name:

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

Test material form:

liquid

Details on test material:

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

Specific details on test material used for the study:

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

Method

Target gene:

Histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) rat liver S9 mix

Test concentrations with justification for top dose:

The following concentrations of the test item were applied in the main experimental phase: ±S9: 5000, 2500, 1600, 500, 160 and 50 µg/plate.
Selection of the concentration range for the main experimental phase (initial mutation test) was done on the basis of the preliminary solubility test and a concentration range finding test (informatory toxicity test). The concentration levels were chosen according to the OECD 471 guideline recommendations. Moreover, at the concentration choice, the noticed mutagenic effect of the test item and the solubility properties (adequately soluble) of the test item were taken into consideration.

Vehicle / solvent:

Vehicle/solvent used: DMSO
Justification for choice of solvent/vehicle: First, the test item was tried to be formulated in ultrapure water. At the concentration of 50 mg/mL the test item was separated from the vehicle as an oil phase. Then DMSO was tested as solvent at 50 mg/mL, and a total dissolution of the test item was observed, and a clear, transparent and homogeneous solution was gained. Since DMSO is one of the preferred vehicles and this vehicle is compatible with the survival of the bacteria and the S9 activity it was chosen as the appropriate solvent of the test item in this study.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive Control concentration 4 µg/plate for TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control concentration: 2 µg/plate for TA100 and TA1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Positive control concentration: 50 µg/plate for TA1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control concentration: 2 µL/plate for WP2 uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive Control concentration with metabolic activation: 2 µg/plate for S. typhimurium strains; 50 µg/plate for E. coli WP2 uvrA

Details on test system and experimental conditions:

TEST SYSTEM
- The study includes a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test) and a main experimental phase consisting of the initial mutation test (plate incorporation test) and the confirmatory mutation test (pre-incubation test, only in case of non-unequivocal results in the initial mutation test). In the preliminary concentration range finding test as well as in the initial mutation test the plate incorporation method was used.
- Origin of the Bacterial Strains: Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were obtained from: Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA. Frozen stock cultures were prepared from the disc cultures.
- Rat Liver S9 Fraction: The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH, Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

METHOD OF APPLICATION
- Initial mutation test: Standard plate incorporation procedure.
- Bacterial cultures: The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-13 hours in a 37°C Benchtop Incubator Shaker.
- Procedure: Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item both in the presence and absence of rat liver S9 as metabolic activation system. Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per controls or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45°C).
The content of the tubes:
* top agar: 2000 µL
* vehicle or solution of test item or positive controls: 100 µL
* overnight culture of test strain: 100 µL
* phosphate buffer (pH: 7.4) or S9 mix: 500 µL
This solution was mixed and poured on the surface of the properly labeled minimal agar plates. For incubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each with the addition of negative and positive controls.
- Exposure duration: The plates were incubated at 37°C for about 48 hours.

NUMBER OF REPLICATES
Triplicates

CONDITIONS FOR THE VALIDITY OF THE TEST
The test (initial mutation tests) is considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers are in the 10^9 cells/mL ordeR.
- The batch of S9 used in this study shows the appropriate biological activity.
- The positive control reference items (known mutagens) show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).
- For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate. For test compounds that are not soluble at 5 mg/plate or 5 µL/plate and that are not toxic at levels lower than an insoluble level, the highest dose tested is at least one insoluble concentration in the final treatment mixture under the actual conditions of the test at the start of the experiment. Insolubility is assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. The test has to include five analyzable concentrations (where the precipitate does not interfere with the scoring) and a minimum of three non precipitated dose levels.

Evaluation criteria:

DETERMINATION OF CYTOTOXICITY
A concentration level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range, and / or
- pinpoint colonies appear, and / or
- reduced background lawn development occurs.

MUTAGENICITY CRITERIA
A result is considered positive if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response: A test article is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

Based on the evaluation criteria no statistical analysis was required.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. typhimurium TA98 and TA1537, and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: S. typhimurium TA1535 and TA100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

VALIDITY CRITERIA
In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled.

CONTROLS
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in all experimental phases of the study. Each of the investigated reference mutagens (positive control) showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases and all revertant numbers fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. The revertant colony numbers of the parallel investigated untreated and ultrapure water (ASTM Type I) control plates in all experimental phases were slightly higher or lower than the DMSO control plates; however, the higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
In summary, the actual values of vehicle, positive and untreated controls were in line with the criteria for validity of the assay.

CYTOTOXICITY RESULTS
In the main mutation test inhibitory effect of the test item was not observed in any of the five examined strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix).

PRECIPITATION
No precipitation of the test item was observed on the plates after about 48 hours incubation in the examined bacterial strains at any examined concentration level (±S9) in the performed main experiment.

MUTAGENICITY RESULTS
Substantial, biologically relevant increases in revertant colony numbers were observed in Salmonella typhimurium TA100 and TA1535 strains following treatment with the test item in the absence and in the presence of exogenous metabolic activation (±S9). The main mutation test results adequately confirmed the positive results of the preliminary concentration range finding test in the case of Salmonella typhimurium TA100 strain.

In Escherichia coli WP2 uvrA at the concentration range of 5000-160 µg/plate in the absence of exogenous metabolic activation (-S9) and at the concentrations of 5000, 2500 and 1600 µg/plate in the presence of exogenous metabolic activation (+S9) the actual mean of revertant colony numbers was above the corresponding historical control data ranges and the actual vehicle control data ranges; but in these cases the increased revertant colony numbers were not above the threshold for being positive (Mutation Rate = 3.00).

In the performed main experiment, in Salmonella typhimurium TA98 and TA1537 occasional increases in the revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, within the corresponding historical control data range and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item Triethylene glycol dimethanesulfonate (CAS No. 80322-82-3) induced gene mutations by base-pair substitutions in the genome of the strains of Salmonella typhimurium TA100 and TA1535 in the absence and presence of exogenous metabolic activation. In conclusion, the test item Triethylene glycol dimethanesulfonate (CAS No. 80322-82-3) has mutagenic activity on two out of five applied bacterium tester strains (Salmonella typhimurium TA100 and TA1535) under the test conditions used in this study.

Executive summary:

Purpose of the study

The test item Triethylene glycol dimethanesulfonate (CAS No. 80322-82-3) was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

Bacterial strains

Salmonella typhimurium TA98, target mutation: hisD3052; mutation type: frameshift;
Salmonella typhimurium TA100, target mutation: hisG46; mutation type: base pair substitution;
Salmonella typhimurium TA1535, target mutation: hisG46; mutation type: base pair substitution;
Salmonella typhimurium TATA1537, target mutation: hisC3076; mutation type: frameshift;
Escherichia coli WP2 uvrA, target mutation: trpE; mutation type: base pair substitution.

 

Exogenous metabolic activation

The experiments were carried out in the presence and absence of a cofactor supplemented post-mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

 

Experimental phases

The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), and a main experimental phase: initial mutation test (plate incorporation test). Because of the obtained unequivocal positive results in the initial mutation test, the performance of a confirmatory mutation test (pre-incubation test) was considered not necessary for final conclusion of the study.

 

Controls

Untreated, negative (vehicle) and strain specific positive controls.

 

Parallels

Three parallels for each concentration level and controls.

 

Vehicle, test item concentrations, rationale for dose selection

Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO) and the following concentrations of the test item were prepared and investigated in the main experimental phase: ±S9: 5000, 2500, 1600, 500, 160 and 50 μg/plate.
At the concentration choice the guideline criterion (OECD 471) for soluble, non-toxic test compounds (where the recommended maximum test concentration is 5000 μg/plate), the noticed mutagenic effect of the test item and the solubility properties (adequately soluble) of the test item were taken into consideration. At the preparation of the test item solutions any correction (multiplier) factor was not taken into consideration, the concentrations were based on the final formulation as is.

 

Validity of the Study

In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean numbers of spontaneous revertants that were in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and all of the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases.

 

Solubility, precipitation

The test item was adequately soluble in dimethyl sulfoxide (DMSO), at 50 mg/mL a clear, transparent and homogeneous solution was obtained.
In the main mutation test (following the plate incorporation procedure), no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

 

Cytotoxicity results

In the main mutation test inhibitory effect of the test item was not observed in any of the five examined strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix).

 

Mutagenicity results

Substantial, biologically relevant increases in revertant colony numbers were observed in Salmonella typhimurium TA100 and TA1535 strains following treatment with the test item in the absence and in the presence of exogenous metabolic activation (±S9). The main mutation test results adequately confirmed the positive results of the preliminary concentration range finding test in the case of Salmonella typhimurium TA100 strain.

 

Conclusion

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item Triethylene glycol dimethanesulfonate (CAS No. 80322-82-3) induced gene mutations by base-pair substitutions in the genome of the strains of Salmonella typhimurium TA100 and TA1535 in the absence and presence of exogenous metabolic activation.

In conclusion, the test item Triethylene glycol dimethanesulfonate (CAS No. 80322-82-3) has mutagenic activity on two out of five applied bacterium tester strains (Salmonella typhimurium TA100 and TA1535) under the test conditions used in this study.