Sodium ethylenesulphonate

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:

1988-03-15 to 1988-03-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

used dose level too low according to current requirements

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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 ethylenesulphonate

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

oxidane

impurity 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 2-hydroxyethanesulfonate

impurity 2

Reference substance name:

Disodium 1,2-ethanedisulphonate

EC Number:

226-198-0

EC Name:

Disodium 1,2-ethanedisulphonate

Cas Number:

5325-43-9

Molecular formula:

C2H6O6S2.2Na

IUPAC Name:

disodium ethane-1,2-disulfonate

impurity 3

Reference substance name:

2,2'-Oxybis-ethanesulfonic acid disodium salt

Cas Number:

63440-92-6

Molecular formula:

C4H8O7S2.2Na

IUPAC Name:

2,2'-Oxybis-ethanesulfonic acid disodium salt

impurity 4

Reference substance name:

sodium 2-sulfonatoethyl sulfate

Molecular formula:

C2H4Na2O7S2

IUPAC Name:

sodium 2-sulfonatoethyl sulfate

impurity 5

Reference substance name:

Sodium sulphate

EC Number:

231-820-9

EC Name:

Sodium sulphate

Cas Number:

7757-82-6

Molecular formula:

H2O4S.2Na

IUPAC Name:

disodium sulfate

impurity 6

Reference substance name:

Sodium hydroxide

EC Number:

215-185-5

EC Name:

Sodium hydroxide

Cas Number:

1310-73-2

Molecular formula:

HNaO

IUPAC Name:

sodium hydroxide

Test material form:

liquid

Method

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:

Type and composition of metabolic activation system:
- source of S9
:
microsomal enzyme system (S-9 mix) from Aroclor 1254 induced Sprague-Dawley rat liver
- method of preparation of S9 mix:
Male Sprague Dawley rats (200 - 300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation was performed at 0 to 4 °C using cold sterile solution and glassware. The livers from at least 5 - 6 animals are removed and pooled, washed in 150 mM KCL (approximately 1 mL/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KCL. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and storage at -80 °C for not longer than three months.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9-Mix

Test concentrations with justification for top dose:

Toxicity test (first and second experiment) and mutagenicity test (first experiment): 0, 4, 20, 100, 500, 2500, 10000 µg/plate of the 30% sodium ethylene sulphonate solution.
Mutagenicity test (second experiment): 0, 4, 20, 100, 500, 2500, 10000 µg/plate of the 30% sodium ethylene sulphonate solution.

Vehicle / solvent:

aqua bidest

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 Mix (TA 100, TA 1535)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 Mix (TA 1537)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 Mix (TA 98, TA 1538)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 Mix (TA 98, TA 100, TA 1535, TA 1537, TA 1538, WPuvrA)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 Mix (TA 98, TA 100, TA 1535, TA 1537, TA 1538, WPuvrA)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Methyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 Mix (WP2uvraA)

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 to 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: thinning of the bacterial lawn

Evaluation criteria:

no data

Statistics:

no data

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

First experiment

Table 1: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 100

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	169	180	178	150
4	-	178	184	188	163
20	-	175	176	172	178
100	-	190	186	204	180
500	-	202	209	202	194
2500	-	185	199	172	185
10000	-	182	185	180	180
0	+	180	185	172	182
4	+	175	185	171	171
20	+	175	166	175	183
100	+	196	202	176	210
500	+	191	200	183	189
2500	+	177	179	182	169
10000	+	179	183	188	167

 

 

Table 2: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1535

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	12	10	12	14
4	-	13	15	10	15
20	-	13	14	15	11
100	-	13	11	12	17
500	-	12	15	12	10
2500	-	14	11	15	16
10000	-	11	11	11	10
0	+	13	15	11	13
4	+	13	14	13	12
20	+	12	13	12	11
100	+	14	17	13	11
500	+	15	11	16	17
2500	+	14	13	14	15
10000	+	13	11	15	12

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Table 3: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1537

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	10	11	8	11
4	-	10	11	9	11
20	-	8	9	7	8
100	-	8	7	9	7
500	-	9	8	9	9
2500	-	8	7	10	7
10000	-	8	9	7	8
0	+	10	10	9	11
4	+	10	10	10	11
20	+	11	11	9	12
100	+	11	10	12	12
500	+	12	11	13	11
2500	+	10	9	13	9
10000	+	10	10	10	11

 

 

Table 4: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1538

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1538

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	12	13	11		13
4	-	13	14	14		12
20	-	13	11	16		12
100	-	14	12	14		15
500	-	15	15	19		11
2500	-	13	15	14		9
10000	-	12	15	11		10
0	+	18	18		16	19
4	+	18	23		16	15
20	+	15	16		14	14
100	+	17	18		16	18
500	+	19	17		20	19
2500	+	18	14		16	21
10000	+	17	19		14	18

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Table 5: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 98

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	25	23	26	25
4	-	26	21	28	28
20	-	25	22	24	28
100	-	23	25	25	20
500	-	23	23	27	20
2500	-	23	20	22	27
10000	-	25	25	28	23
0	+	30	29	32	29
4	+	34	34	35	33
20	+	31	33	32	29
100	+	33	30	38	32
500	+	35	34	32	38
2500	+	35	32	35	37
10000	+	32	31	34	30

 

 

Table 6: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

WP2uvrA

Number of revertant colonies per plate and mean values using Escherichia coli strain WP2uvrA

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	40	45	39		35
4	-	38	39	40		35
20	-	40	40	40		39
100	-	37	37	36		37
500	-	42	42	43		40
2500	-	41	43	37		42
10000	-	40	42	40		37
0	+	44	48		36	48
4	+	42	42		45	39
20	+	45	42		51	42
100	+	45	41		48	45
500	+	39	41		39	38
2500	+	42	43		43	39
10000	+	43	36		46	46

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Second experiment

Table 7: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 100

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate			Surviving fraction
0	-	143	152	143	133	1.0
4	-	147	150	136	154	1.0
20	-	142	143	119	164	1.0
100	-	156	161	150	157	1.0
500	-	148	158	119	168	1.0
2500	-	141	129	127	167	0.9
5000	-	142	140	126	160	1.0
0	+	183	183	195	172	1.0
4	+	165	155	158	182	1.0
20	+	174	181	158	182	1.0
100	+	178	197	163	173	0.9
500	+	155	187	155	154	1.0
2500	+	168	191	159	153	0.9
5000	+	172	200	158	159	0.9

 

 

Table 8: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1535

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	13	13	12		14
4	-	13	12	14		14
20	-	12	12	10		14
100	-	12	14	10		11
500	-	14	13	11		17
2500	-	11	10	12		10
5000	-	11	9	14		11
0	+	12	12		12	13
4	+	12	10		13	13
20	+	13	11		11	16
100	+	13	11		15	14
500	+	12	10		13	12
2500	+	12	13		15	9
5000	+	11	12		11	10

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Table 9: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1537

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	7	9	7	6
4	-	8	7	8	8
20	-	7	5	9	6
100	-	7	6	8	8
500	-	7	5	7	8
2500	-	7	6	9	6
5000	-	7	9	6	7
0	+	9	10	8	8
4	+	9	9	10	7
20	+	10	10	10	11
100	+	10	8	12	11
500	+	8	7	9	9
2500	+	9	9	9	8
5000	+	10	10	10	9

 

 

Table 10: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1538

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1538

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	12	12	11		13
4	-	12	13	12		10
20	-	14	15	15		13
100	-	12	13	13		12
500	-	11	9	11		13
2500	-	14	14	14		15
5000	-	12	10	11		14
0	+	19	16		21	20
4	+	17	15		17	19
20	+	19	17		20	20
100	+	17	18		19	15
500	+	17	17		18	15
2500	+	19	17		22	17
5000	+	17	15		18	18

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Table 11: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 98

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose µg/plate	Metabolic activation	Mean value22	Colonies per plate
0	-	22	23	22	22
4	-	22	20	21	25
20	-	27	26	27	27
100	-	23	24	24	20
500	-	21	24	19	19
2500	-	24	30	20	21
5000	-	22	21	22	24
0	+	25	23	26	27
4	+	29	28	28	32
20	+	28	30	27	27
100	+	26	30	20	27
500	+	28	31	25	29
2500	+	27	23	31	27
5000	+	25	23	28	25

 

 

Table 12: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

WP2uvrA

Number of revertant colonies per plate and mean values using Escherichia coli strain WP2uvrA

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	51	52	56		45
4	-	50	43	46		60
20	-	54	54	56		53
100	-	51	52	49		51
500	-	52	51	55		50
2500	-	53	51	57		50
5000	-	57	54	61		55
0	+	56	54		56	59
4	+	58	52		61	62
20	+	54	53		54	54
100	+	56	54		53	60
500	+	59	59		60	58
2500	+	57	56		60	56
5000	+	53	52		49	58

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

First experiment

Table 13: mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain	Compound	Dose µg/plate	Metab. Activ.	Mean Value	Colonies per plate
TA100	Sodium-azide	1	-	526	456	549	574
TA1535	Sodium-azide	1	-	409	410	398	419
TA1537	9-Aminoacridine	50	-	95	81	102	103
TA1538	2-Nitrofluorene	2.5	-	462	432	487	467
TA98	2-Nitrofluorene	2.5	-	327	294	355	333
WP2uvrA	MNNG	1.5	-	261	285	256	243
-	Vinylsulfon- saures Natrium 30%ig	10000	-	0	0	0	0

- : absence

 

 

Table 13a : mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain	Compound	Dose ug/plate	Metab. Activ.	Mean Value	Colonies per plate
TA100	2-Aminoanthracen	0.5	+	818	874	806	775
TA1535	2-Aminoanthracen	1	+	140	151	126	142
TA1537	2-Aminoanthracen	1	+	126	122	130	125
TA1538	2-Aminoanthracen	0.5	+	458	438	463	473
TA98	2-Aminoanthracen	0.5	+	426	407	444	426
WP2uvrA	2-Aminoanthracen	10	+	440	428	442	451
TA100	Benzo[a]pyrene	10	+	1311	1352	1298	1283
TA1535	Benzo[a]pyrene	10	+	18	15	18	21
TA1537	Benzo[a]pyrene	10	+	96	85	100	102
TA1538	Benzo[a]pyrene	10	+	176	206	173	149
TA98	Benzo[a]pyrene	10	+	550	535	585	531
WP2uvrA	Benzo[a]pyrene	10	+	87	97	75	88
-	S-9 mix	500 µl	+	0	0	0	0
-	Vinylsulfon- saures Natrium 30%ig	1000 µg	+	0	0	0	0

+ :presence

 

 

Second experiment

Table 14: mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain	Compound	Dose µg/plate	Metab. Activ.	Mean Value	Colonies per plate
TA100	Sodium-azide	1	-	293	29+2	306	280
TA1535	Sodium-azide	1	-	272	277	287	252
TA1537	9-Aminoacridine	50	-	157	160	163	178
TA1538	2-Nitrofluorene	2.5	-	459	469	430	478
TA98	2-Nitrofluorene	2.5	-	340	314	379	328
WP2uvrA	MNNG	2.5	-	223	206	223	241
-	Vinylsulfon- saures Natrium 30%ig	5000	-	0	0	0	0

- : absence

 

 

Table 14a : mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain	Compound	Dose µg/plate	Metab. Activ.	Mean Value	Colonies per plate
TA100	2-Aminoanthracen	0.5	+	465	399	497	502
TA1535	2-Aminoanthracen	1	+	88	77	84	103
TA1537	2-Aminoanthracen	1	+	90	79	108	84
TA1538	2-Aminoanthracen	0.5	+	494	474	549	458
TA98	2-Aminoanthracen	0.5	+	577	589	563	579
WP2uvrA	2-Aminoanthracen	10	+	481	463	496	483
TA100	Benzo[a]pyrene	10	+	685	746	670	639
TA1535	Benzo[a]pyrene	10	+	18	16	15	24
TA1537	Benzo[a]pyrene	10	+	86	81	96	80
TA1538	Benzo[a]pyrene	10	+	183	176	184	188
TA98	Benzo[a]pyrene	10	+	551	552	547	583
WP2uvrA	Benzo[a]pyrene	10	+	74	66	79	76
-	S-9 mix	500 ul	+	0	0	0	0
-	Vinylsulfon- saures Natrium 30%ig	5000 ug	+	0	0	0	0

+ :presence

 

Applicant's summary and conclusion

Conclusions:

The test solution, containing 30% sodium ethylene sulphonate, did not demonstrate mutagenic potential. Under the conditions of the present bacterial reverse mutation assay the maximum test concentration of sodium ethylene sulphonate was 3000 µg/plate.

Executive summary:

Sodium ethylene sulphonate was tested as a 30% aqueous solution in the Salmonella typhimurium reverse mutation assay (Hoechst AG (b), 1988) with the strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) similar to OECD guideline 471 and GLP. Two independent mutagenicity studies were conducted (plate incorporation method), each in the absence and in the presence of a metabolising system derived from a rat liver homogenate. For both studies each bacterial strain was exposed to 6 dose levels. In the first experiment concentrations ranged from 4 to 10000 µg/plate and in the second experiment from 4 to 5000 µg/plate.Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test solution proved to be not toxic to the bacterial strains. 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 the 30% sodium ethylene sulphonate solution did not result in relevant increases in the number of revertant colonies.In conclusion the test solution, containing 30% sodium ethylene sulphonate, did not demonstrate mutagenic potential. Under the conditions of the present bacterial reverse mutation assay the maximum test concentration of sodium ethylene sulphonate was 3000 µg/plate.