Sodium 2-hydroxyethanesulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-08-19 to 1992-08-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-hydroxyethanesulphonate

EC Number:

216-343-6

EC Name:

Sodium 2-hydroxyethanesulphonate

Cas Number:

1562-00-1

Molecular formula:

C2H6O4S.Na

IUPAC Name:

sodium 1,4-bis[(2-ethylhexyl)oxy]-1,4-dioxobutane-2-sulfonate

Details on test material:

- Name of test material (as cited in study report): Ethansalz 97/100 %
- Molecular weight: 148.05 g/mol
- Chemical nomenclature: Ethansulfonacid, 2-Hydroxy-, Monosodiumsalt
- Substance type: solid
- Physical state: white powder
- Analytical purity: 99.6%
- Purity test date: 1992-01-21
- Lot/batch No.: 95
- Expiration date of the lot/batch: 1994-01-21
- Stability under test conditions: stable
- Storage condition of test material: dark, at room temperature
- melting point: appr. 230°C

Method

Species / strain

Species / strain 1

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Test item:
- Toxicity test: 4 - 10000 µg/plate in aqua bidest.
- Mutagenicity test: 4-5000 µg/plate in aqua bidest.

Positive controls:
- sodium azide: 1 µg/plate
- 9-Aminoacridine: 50 µg/plate
- 2-Nitrofluorene: 2.5 µg/plate
- N-Methyl-N-nitro-N-nitrosoguanidine: 2.5 µg/plate
- 2-Aminoanthracen: 0.5-10 µg/plate

Vehicle / solvent:

Aqua bidest.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: for details on controls please see below

Details on test system and experimental conditions:

The assay was performed in two independent experiments (3 plates/dose, with and without metabolic activation):

1. Experiment: 0-10000 µg test item/plate
2. Experiment: 0-5000 µg test item/plate

DURATION:
After mixing the overnight culture and test compound the mixture is poured into a petridish with minimal agar and incubated for approx. 48h at 37°C in the dark.

The first experiment was performed with all tester strains using three plates per dose to get information on mutagenicity and cytotoxicity for calculation of an appropriate dose range. A reduced rate of spontanously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity.

POSITIVE CONTROL SUBSTANCES:
without metabolic activation: Sodium azide (TA 100, TA 1535), 9-Aminoacridine (TA 1537), 2-Nitrofluorene (TA 98, TA 1538), N-Methyl-N-nitro-N-nitrosoguanidine (WP2uvrA)
with metabolic activiation: 2-Aminoanthracene (TA 98, TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA)

Evaluation criteria:

A test article is classified mutagenic if either of the following conditions under a) and b) is achieved:
a) a test article produced at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn.
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean numberof revertants per plate of the appropriate verhicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The tests must be reproducible.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1538

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:

valid

Positive controls validity:

valid

Test results 2

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Ames Test: Ethansalz 97/100 (Study # 92.0810)

 

 

Table: Summary of mean number of revertants (mean of 3 plates) in Salmonella typhimurium andE. coli strains with and without metabolic activation

Experiment I
Concentration	TA 98				TA 100				TA1535		TA 1537
[µg/plate]	- S9 mix		+ S9 mix		- S9 mix		+ S9 mix		- S9 mix	+ S9 mix	- S9 mix	+ S9 mix
0	24		31		151		187		13	13	9	12
4	27		33		153		185		10	14	9	9
20	25		35		156		189		10	15	9	9
100	26		34		155		184		12	13	9	11
500	26		33		150		187		15	12	9	11
2500	29		30		161		181		12	13	9	12
10000	31		34		161		184		12	16	7	10
Experiment I
		TA 1538				WP2uvrA
		- S9 mix		+ S9 mix		- S9 mix		+ S9 mix
0		20		27		29		31
4		19		23		28		31
20		19		25		30		26
100		21		27		29		28
500		20		27		32		31
2500		21		24		30		30
10000		18		23		29		30
Experiment II
		TA 98				TA 100			TA1535		TA 1537
		- S9 mix		+ S9 mix		- S9 mix		+ S9 mix	- S9 mix	+ S9 mix	- S9 mix	+ S9 mix
0		24		25		134		134	13	14	13	12
4		26		22		139		140	10	13	13	14
20		26		24		133		137	8	15	12	12
100		21		23		148		141	8	12	15	15
500		22		25		153		155	10	11	14	14
2500		18		31		151		153	10	10	14	11
5000		25		24		156		156	12	14	15	12
Experiment II
		TA 1538				WP2uvrA
		- S9 mix		+ S9 mix		- S9 mix		+ S9 mix
0		22		22		29		31
4		20		19		32		37
20		17		24		35		39
100		19		24		30		36
500		19		20		25		33
2500		19		21		28		39
5000		19		17		29		34

 

The sensitivity of the test system used was demonstrated by the induction of an increased number of revertants by the positive controls.

Applicant's summary and conclusion

Conclusions:

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or presence of S9 mix. No dose dependent effect was obtained.

it is concluded that the test substance is not mutagenic in these bacterial test systems either in the absence or in the presence of an exogenous metabolizing system.

Executive summary:

Ethansalz 97/100 % was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and E. coli WP2uvrA.

The mutagenicity studies were conducted in the absence and presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 µg/plate to 10000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positve control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Ethansalz 97/100 % did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that Ethansalz 97/100 % is not mutagenic in these bacterial test sytems either with or without exogenous metabolic activation at the dose levels investigated.