Sodium 2-bromoethanesulphonate

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:

06 September 2016 - 08 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-bromoethanesulphonate

EC Number:

224-244-4

EC Name:

Sodium 2-bromoethanesulphonate

Cas Number:

4263-52-9

Molecular formula:

C2H5BrO3S.Na

IUPAC Name:

sodium 2-bromoethanesulphonate

Test material form:

solid

Method

Target gene:

his/trp

Species / strain

Species / strain / cell type:

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

Remarks:

uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor 1254 induced male Wistar rat liver

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system. A maximum concentration of 5000 µg/plate was selected, in order that initial treatments were performed up to the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).
1st series: 5.0, 15.8, 50, 158, 500, 1580, 5000 µg/plate, with or without 10% S9 mix
2nd series: 50, 500, 1580, 2810, 5000 µg/plate, with or without 30% S9 mix

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Solubility properties of the test item, non-toxicity to bacteria.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: Daunomycin DAUN, 1 µg/plate; 2-Aminoanthracene 2-AA, 2.0, 5.0, 10.0 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 -3 days

SELECTION AGENT: via medium

NUMBER OF REPLICATIONS: 2, 3 parallel plates were used for each concentration step of the lest material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.

DETERMINATION OF CYTOTOXICITY
- Method: The presence of a background lawn of non-reverlant cells was checked for each plate.

Evaluation criteria:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established. All further results, ranging between "no" and "clear", are assessed as "weak increases".

Interpretations:
A test material was to be defined as negative or non-mutagenic in this assay if
• the assay was to be considered valid, and
• "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)
For valid data, the test material was considered to be positive or mutagenic if:
• a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
• "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.
In general two series of experiments must be performed. However, there was no requirement for verification of a clear positive response.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Results 1st series:

Metabolic Activation	Test Material	Conc. [µg/plate]	Revertants per plate (mean ± SD)
			TA98	TA100	TA1535	TA1537	E.coli
Without Activation	H2O		34 ± 4	117 ± 11	29 ± 3	31 ± 8	37 ± 7
	Test substance	5.0	36 ± 10	119 ± 23	26 ± 7	28 ± 6	39 ± 3
		15.8	28 ± 7	114 ± 11	28 ± 5	29 ± 6	31 ± 7
		50.0	36 ± 6	121 ± 17	27 ± 10	31 ± 1	43 ± 16
		158	31 ± 6	112 ± 11	29 ± 8	32 ± 4	38 ± 6
		500	23 ± 2	126 ± 9	26 ± 7	30 ± 6	31 ± 5
		1580	26 ± 6	120 ± 11	26 ± 3	35 ± 7	37 ± 8
		5000	31 ± 3	106 ± 8	31 ± 4	30 ± 4	38 ± 9
	DAUN	1.0	360 ± 9
	NaN3	2.0		1505 ± 132	974 ± 36
	9-AA	50.0				783 ± 317
	NQO	2.0					1769 ± 101
With Activation	H2O		34 ± 8	148 ± 12	28 ± 7	35 ± 1	45 ± 5
	Test Substance	5.0	30 ± 2	137 ± 4	20 ± 3	35 ± 6	56 ± 4
		15.8	39 ± 3	144 ± 15	21 ± 1	30 ± 5	39 ± 12
		50.0	34 ± 10	136 ± 7	29 ± 12	27 ± 1	44 ± 1
		158	35 ± 4	134 ± 11	32 ± 6	28 ± 8	41 ± 4
		500	37 ± 1	141 ± 14	33 ± 11	29 ± 6	46 ± 7
		1580	29 ± 3	134 ± 14	37 ± 3	34 ± 1	41 ± 2
		5000	34 ± 13	148 ± 6	25 ± 3	29 ± 4	38 ± 4
	2-AA	2.0	340 ± 10	1178 ± 103
		5.0			245 ± 20	415 ± 33
		10.0					378 ± 15

Results 2nd series:

Metabolic Activation	Test Material	Conc. [µg/plate]	Revertants per plate (mean ± SD)
			TA98	TA100	TA1535	TA1537	E.coli
Without Activation	H2O		24 ± 6	132 ± 8	26 ± 8	28 ± 4	35 ± 11
	Test substance	50.0	27 ± 4	122 ± 10	34 ± 9	30 ± 6	32 ± 10
		500	30 ± 8	120 ± 22	29 ± 8	26 ± 5	33 ± 4
		1580	28 ± 6	122 ± 14	28 ± 6	28 ± 2	40 ± 3
		2810	34 ± 5	136 ± 11	28 ± 4	28 ± 7	28 ± 4
		5000	23 ± 5	115 ± 5	32 ± 5	26 ± 10	32 ± 5
	DAUN	1.0	159 ± 36
	NaN3	2.0		1537 ± 76	927 ± 41
	9-AA	50.0				1482 ± 257
	NQO	2.0					1741 ± 112
With Activation	H2O		31 ± 5	137 ± 16	28 ± 7	32 ± 8	40 ± 9
	Test Substance	50.0	27 ± 7	135 ± 11	21 ± 3	35 ± 1	45 ± 17
		500	22 ± 2	149 ± 6	29 ± 5	37 ± 8	36 ± 12
		1580	28 ± 9	125 ± 22	24 ± 4	36 ± 3	42 ± 3
		2810	29 ± 6	131 ± 21	31 ± 2	39 ± 4	42 ± 3
		5000	22 ± 6	131 ± 2	23 ± 2	33 ± 6	37 ± 2
	2-AA	2.0	176 ± 8
		5.0		19970 ± 61
		10.0			164 ± 5	390 ± 13	181 ± 9

Applicant's summary and conclusion

Conclusions:

In an in vitro bacterial reverse mutagenicity assay (AMES) according to OECD Guideline 471, the test item did not show a mutagenic potential.

Executive summary:

In an in vitro bacterial reverse mutagenicity assay (AMES) according to OECD Guideline 471, the mutagenic potential of the test item was determined. The assay was performed in Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated male Wistar rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10% S9 mix were used in the 1st (test item: 5.0, 15.8, 50, 158, 500, 1580, 5000 µg/plate), and 30% S9 in the 2nd series (test item: 50, 500, 1580, 2810, 5000 µg/plate), respectively.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following test substance treatments of all the tester strains in the absence and presence of S9 mix, no relevant increases in revertanl numbers were observed. It is concluded that with and without addition of S9 mix the test substance was not mutagenic under the experimental conditions.