Benzenesulphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

Benzenesulphonic acid

EC Number:

202-638-7

EC Name:

Benzenesulphonic acid

Cas Number:

98-11-3

Molecular formula:

C6H6O3S

IUPAC Name:

benzenesulfonic acid

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

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

Remarks:

Permanent stocks of these strains are kept at -80°C in ERBC facilities.

Metabolic activation:

with and without

Metabolic activation system:

One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, were used in this study and had the following characteristics:
Species Rat
Strain Sprague Dawley
Tissue Liver
Inducing Agents Phenobarbital – 5,6-Benzoflavone
Producer MOLTOX,Molecular Toxicology, Inc.
Batch Number 4086 (Main Assays)

The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL):
S9 tissue fraction 1.0mL
NADP (100 mM) 0.4mL
G-6-P (100 mM) 0.5mL
KCl (330 mM) 1.0mL
MgCl2 (100 mM) 0.8mL
Phosphate buffer (pH 7.4, 200 mM) 5.0mL
DistilledWater 1.3mL

Test concentrations with justification for top dose:

Toxicity test: 5, 1,58, 0,5, 0,158 and 0,05 µL/plate
I Main assay:
TA1535 without metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA1535 with metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA1537 without metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA1537 with metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA98, TA100 and WP2 uvrA with and without metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 and 0.156 μL/plate

II Main assay:
TA1535 without metabolic activation: 2.5, 1,25, 0,625, 0.313, 0.156, 0,0781 μL/plate
TA1535 with metabolic activation: 2.5, 1,25, 0,625, 0.313, 0.156, 0,0781 μL/plate
TA1537 without metabolic activation: 2.5, 1,25, 0,625, 0.313, 0.156, 0,0781 μL/plate
TA1537 with metabolic activation: 2.5, 1,25, 0,625, 0.313, 0.156 0,0781 μL/plate
TA100 without metabolic activation: 2.5, 1,25, 0,625, 0.313, 0.156, 0,0781 μL/plate
TA100 with metabolic activation: 2.5, 1,25, 0,625, 0.313, 0.156, 0,0781 μL/plate
TA98 and WP2 uvrA with and without metabolic activation: 2.00, 1.00, 0.5, 0.250, 0.125, 0.0625 μL/plate

Vehicle / solvent:

Water for injection

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100 wihtout metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.Coli WP2 uvrA wihtout metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with metabolic activation

Details on test system and experimental conditions:

Solubility of the test item was evaluated in a preliminary trial using sterile water for injection. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found soluble at 50.0 μL/mL (expressed as active
ingredient). This result permitted a maximum concentration of 5.00 μL/plate to be used in the toxicity test.

The Main Assay I was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.

The overlay mixture was composed as follows:
Overlay agar (held at 45°C) 2.0mL
Test or control item solution 0.1mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
Bacterial suspension 0.1mL

The Main Assay II was performed using a pre-incubation method. The components were added in turn to an empty test-tube:
Bacterial suspension 0.1mL
Test item solution 0.1mL
or control item solution 0.05mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL

The incubate was vortexed and placed at 37°C for 30 minutes. 2 mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were immediately scored by counting the number of revertant colonies in each plate. In main assay II, plates were held at 4°C for 24 hours before scoring.

Permanent stocks of tested strains are kept at -80°C in ERBC.Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures,
which had been checked for the presence of the appropriate genetic markers.

Evaluation criteria:

Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study.
TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens. In addition to amutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.
Tester strain WP2 uvrA is reverted fromtryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.

The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
No precipitation of the test item was observed at the end of the incubation period at any concentration tested, in the absence or presence of S9 metabolic activation.
Moderate to slight toxicity was seen at the two highest concentrations with WP2 uvrA, both in the absence and presence of S9 metabolic activation. A less pronounced toxic effect was noticed with the remaining tester strains, as indicated by slight reduction in revertant
numbers and/or thinning of the background lawn at the highest dose level, in the absence and presence of S9 metabolism

I Main assay: No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.
Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at the two highest dose levels both in the absence and presence of S9 metabolism, with TA1535, TA1537, TA98 and TA100 tester strains. Moderate to severe reduction of the background lawn was seen with WP2 uvrA tester strain, at the two highest dose levels,both in the absence and presence of S9 metabolism.
II Main assay: Toxicity was observed at the highest dose level both in the absence and presence of S9 metabolism, with all tester strains.
In both main tests, the test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Controls: Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.

Controls of S9:
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Any other information on results incl. tables

Citoxicity and mutagenicity results are given in the following tables

BBL: Bacterial background lawn

A3847: Cytotoxicity - plate incorporation test without metabolic activation
Test item (μL per plate)	TA98	TA100	TA1535	TA1537	E.Coli wp2 UVRa	Bacteriotoxic effect	Cytotoxicity
Untreated	31	124	15	17	26	not evaluated	no cytotoxicity
0.0500	28	117	15	15	24	BBL: normal	no cytotoxicity
0.158	29	120	15	14	29	BBL: normal	no cytotoxicity
0.500	35	133	16	17	23	BBL: normal	no cytotoxicity
1.58	25	125	17	18	29	BBL: slightly thinned (WP2uvrA)	Slight (WP2uvrA)
5.00	23	102	13	17	30	BBL: slightly/moderately thinned	Slight/moderate
Rt/Rc 50	0,90	0,94	1,00	0,88	0,92
Rt/Rc 158	0,94	0,97	1,00	0,82	1,12
Rt/Rc 500	1,13	1,07	1,07	1,00	0,88
Rt/Rc 1580	0,81	1,01	1,13	1,06	1,12
Rt/Rc 5000	0,74	0,82	0,87	1,00	1,15
A3847: Cytotoxicity - plate incorporation test with metabolic activation
Test item (μL per plate)	TA98	TA100	TA1535	TA1537	E.Coli wp2 UVRa	Bacteriotoxic effect	Cytotoxicity
Untreated	41	125	20	21	32	not evaluated	no cytotoxicity
0.0500	39	125	14	23	32	BBL: normal	no cytotoxicity
0.158	38	122	15	25	28	BBL: normal	no cytotoxicity
0.500	35	126	14	20	31	BBL: normal	no cytotoxicity
1.58	35	133	14	18	31	BBL: slightly thinned (WP2uvrA)	Slight (WP2uvrA)
5.00	32	105	15	17	34	BBL: slightly/moderately thinned	Slight/moderate
Rt/Rc 50	0,95	1,00	0,70	1,10	1,00
Rt/Rc 158	0,93	0,98	0,75	1,19	0,88
Rt/Rc 500	0,85	1,01	0,70	0,95	0,97
Rt/Rc 1580	0,85	1,06	0,70	0,86	0,97
Rt/Rc 5000	0,78	0,84	0,75	0,81	1,06

A3847 Μutagenicity Experiment I - plate incorporation test - Strain S. typhimurium TA 98
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	33	32	30	32	0,9	-	not evaluated	no cytotoxicity	46	42	41	43	1,5	-	not evaluated	no cytotoxicity	valid
0.156	35	39	40	38	1,5	1,19	BBL: normal	no cytotoxicity	41	44	37	41	2	0,95	BBL: normal	no cytotoxicity	not mutagenic
0.313	39	35	29	34	2,9	1,06	BBL: normal	no cytotoxicity	34	42	43	40	2,8	0,93	BBL: normal	no cytotoxicity
0.625	40	31	26	32	4,1	1,00	BBL: normal	no cytotoxicity	40	44	39	41	1,5	0,95	BBL: normal	no cytotoxicity
1.25	35	39	34	36	1,5	1,13	BBL: normal	no cytotoxicity	33	39	39	37	2	0,86	BBL: normal	no cytotoxicity
2.50	34	41	36	37	2,1	1,16	BBL: moderately thinned	Moderate	40	35	26	34	4,1	0,79	BBL: moderately thinned	Moderate
5.00	28	29	30	29	0,6	0,91	BBL: moderately thinned	Moderate	39	34	28	34	3,2	0,79	BBL: moderately thinned	Moderate
2-Nitrofluorene/2-AA	165	169	171	168	1,8	5,42			531	521	522	525	3,2	12,21			valid
DMSO	30	30	34	31	1,3	-			46	44	41	44	1,5	-			valid
A3847 Μutagenicity Experiment I - plate incorporation test - Strain S. typhimurium TA 100
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	181	185	170	179	4,5	-	not evaluated	no cytotoxicity	179	180	181	180	0,6	-	not evaluated	no cytotoxicity	valid
0.156	172	170	169	170	0,9	0,95	BBL: normal	no cytotoxicity	184	187	179	183	2,3	1,02	BBL: normal	no cytotoxicity	not mutagenic
0.313	160	164	169	164	2,6	0,92	BBL: normal	no cytotoxicity	184	182	181	182	0,9	1,01	BBL: normal	no cytotoxicity
0.625	164	168	170	167	1,8	0,93	BBL: normal	no cytotoxicity	173	171	175	173	1,2	0,96	BBL: normal	no cytotoxicity
1.25	145	147	149	147	1,2	0,82	BBL: normal	no cytotoxicity	158	160	162	160	1,2	0,89	BBL: normal	no cytotoxicity
2.50	124	117	119	120	2,1	0,67	BBL: slightly thinned	Slight	140	139	139	139	0,3	0,77	BBL: slightly thinned	Slight
5.00	100	89	93	94	3,2	0,53	BBL: moderately thinned / RRN	Moderate	96	93	95	95	0,9	0,53	BBL: moderately thinned / RRN	Moderate
AS/2AA	574	540	533	549	12,7	3,07			1021	1203	1311	1178	84,6	6,54			valid
DMSO						-			169	171	173	171	1,2	-			valid
A3847 Μutagenicity Experiment I - plate incorporation test - Strain S. typhimurium TA 1535
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	12	13	13	13	0,3	-	not evaluated	no cytotoxicity	12	15	16	14	1,2	-	not evaluated	no cytotoxicity	valid
0.313	17	20	15	17	1,5	1,31	BBL: normal	no cytotoxicity	18	20	14	17	1,8	1,21	BBL: normal	no cytotoxicity	not mutagenic
0.625	18	16	13	16	1,5	1,23	BBL: normal	no cytotoxicity	15	18	12	15	1,7	1,07	BBL: normal	no cytotoxicity
1.25	12	13	20	15	2,5	1,15	BBL: normal	no cytotoxicity	20	18	17	18	0,9	1,29	BBL: normal	no cytotoxicity
2.50	12	12	11	12	0,3	0,92	BBL: slightly thinned	Slight	17	17	15	16	0,7	1,14	BBL: slightly thinned	Slight
5.00	14	10	8	11	1,8	0,85	BBL: moderately thinned / RRN	Moderate	15	16	15	15	0,3	1,07	BBL: moderately thinned	Moderate
AS/2-AA	489	512	482	494	9,1	38,00			154	157	149	153	2,3	10,20			valid
DMSO						-			15	17	13	15	2,3	-			valid
A3847 Μutagenicity Experiment I - plate incorporation test - Strain S. typhimurium TA 1537
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	20	19	18	19	0,6	-	not evaluated	no cytotoxicity	20	20	29	23	3	-	not evaluated	no cytotoxicity	valid
0.313	12	16	16	15	1,3	0,89	BBL: normal	no cytotoxicity	12	21	15	16	2,6	0,70	BBL: normal	no cytotoxicity	not mutagenic
0.625	13	19	17	16	1,8	0,94	BBL: normal	no cytotoxicity	15	21	14	17	2,2	0,74	BBL: normal	no cytotoxicity
1.25	13	15	21	16	2,4	1,17	BBL: normal	no cytotoxicity	25	18	21	21	2	0,91	BBL: normal	no cytotoxicity
2.50	12	12	9	11	1	0,50	BBL: slightly thinned / RRN	Slight	20	18	21	20	0,9	0,87	BBL: slightly thinned	Slight
5.00	13	8	7	9	1,9	0,39	BBL: moderately thinned / RRN	Moderate	21	18	17	19	1,2	0,83	BBL: moderately thinned	Moderate
9-AAc/2-AA	172	121	153	149	14,9	9,56			99	101	103	101	1,2	4,59			valid
DMSO	12	17	16	15	1,5	-			25	19	21	22	1,8	-			valid
A3847 Mutagenicity Experiment I - plate incorporation test - Strain E. coli WP2 uvrA
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	22	25	24	24	0,9	-	not evaluated	no cytotoxicity	26	33	30	30	2	-	not evaluated	no cytotoxicity	valid
0.156	29	27	29	28	0,7	1,21	BBL: normal	no cytotoxicity	30	35	33	33	1,5	1,10	BBL: normal	no cytotoxicity	not mutagenic
0.313	22	17	28	22	3,2	1,17	BBL: normal	no cytotoxicity	43	37	32	37	3,2	1,23	BBL: normal	no cytotoxicity
0.625	25	27	25	26	0,7	1,04	BBL: normal	no cytotoxicity	35	31	35	34	1,3	1,13	BBL: normal	no cytotoxicity
1.25	28	25	23	25	1,5	0,96	BBL: normal	no cytotoxicity	30	30	26	29	1,3	0,97	BBL: normal	no cytotoxicity
2.50	23	29	24	25	1,9	1,00	BBL: moderately thinned	Moderate	30	31	30	30	0,3	1,00	BBL: moderately thinned	Moderate
5.00	25	18	23	22	2,1	0,96	BBL: severely thinned	Severe	25	29	23	26	1,8	0,87	BBL: severely thinned	Severe
MMS/2-AA	151	153	155	153	1,2	6,46			191	197	214	201	6,9	5,91			valid
DMSO						-			36	35	30	34	1,9	-			valid

A3847 Μutagenicity Experiment II - pre-incubation test - Strain S. typhimurium TA 98
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	28	36	35	33	2.5	-	not evaluated	no cytotoxicity	41	38	34	38	2	-	not evaluated	no cytotoxicity	valid
0.0625	29	27	24	27	1,5	0,82	BBL: normal	no cytotoxicity	31	37	32	33	1,9	0,87	BBL: normal	no cytotoxicity	not mutagenic
0.125	23	23	28	25	1,7	0,76	BBL: normal	no cytotoxicity	35	35	35	35	0	0,92	BBL: normal	no cytotoxicity
0.250	32	31	24	29	2,5	0,88	BBL: normal	no cytotoxicity	39	38	44	40	1,9	1,05	BBL: normal	no cytotoxicity
0.500	28	29	31	29	0,9	0,88	BBL: normal	no cytotoxicity	38	34	33	35	1,5	0,92	BBL: normal	no cytotoxicity
1.00	27	26	29	27	0,9	0,82	BBL: normal	no cytotoxicity	31	35	36	34	1,5	0,89	BBL: normal	no cytotoxicity
2.00	26	35	24	28	3,4	0,85	BBL: slightly thinned	Slight	38	35	32	35	1,7	0,92	BBL: slightly thinned	Slight
2-Nitrofluorene/2-AA	184	171	196	184	7,2	6,13			615	663	591	623	21,2	16,39			valid
DMSO	33	26	32	30	2,2	-			33	35	37	35	1,2	-			valid
A3847 Μutagenicity Experiment II - pre-incubation test - Strain S. typhimurium TA 100
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	121	123	138	127	5,4	-	not evaluated	no cytotoxicity	134	141	134	136	2,3	-	not evaluated	no cytotoxicity	valid
0.0781	129	122	121	124	2,5	0,98	BBL: normal	no cytotoxicity	125	131	134	130	2,6	0,96	BBL: normal	no cytotoxicity	not mutagenic
0.156	121	143	139	134	6,8	1,06	BBL: normal	no cytotoxicity	126	137	132	132	3,2	0,97	BBL: normal	no cytotoxicity
0.313	139	122	124	128	5,4	1,01	BBL: normal	no cytotoxicity	119	122	138	126	5,9	0,93	BBL: normal	no cytotoxicity
0.625	137	146	129	137	4,9	1,08	BBL: normal	no cytotoxicity	136	145	124	135	6,1	0,99	BBL: normal	no cytotoxicity
1.25	131	132	124	129	2,5	1,02	BBL: normal	no cytotoxicity	127	138	141	135	4,3	0,99	BBL: normal	no cytotoxicity
2.50	124	130	139	131	4,4	1,03	BBL: slightly thinned	Slight	136	124	142	134	5,3	0,99	BBL: slightly thinned	Slight
AS/2AA	766	619	603	663	51,9	5,22			1105	974	1361	1147	113,6	8,43			valid
DMSO						-			120	128	138	129	5,2	-			valid
A3847 Μutagenicity Experiment II - pre-incubation test - Strain S. typhimurium TA 1535
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	19	14	15	16	1,5	-	not evaluated	no cytotoxicity	14	17	16	16	0,9	-	not evaluated	no cytotoxicity	valid
0.0781	13	18	16	16	1,5	1,00	BBL: normal	no cytotoxicity	17	18	16	17	0,6	1,06	BBL: normal	no cytotoxicity	not mutagenic
0.156	19	17	14	17	1,5	1,06	BBL: normal	no cytotoxicity	15	14	15	15	0,3	0,94	BBL: normal	no cytotoxicity
0.313	13	14	16	14	0,9	0,88	BBL: normal	no cytotoxicity	20	17	16	18	1,2	1,13	BBL: normal	no cytotoxicity
0.625	19	18	14	17	1,5	1,06	BBL: normal	no cytotoxicity	21	17	18	19	1,2	1,19	BBL: normal	no cytotoxicity
1.25	14	14	16	15	0,7	0,94	BBL: normal	no cytotoxicity	19	16	18	18	0,9	1,13	BBL: normal	no cytotoxicity
2.50	17	16	13	15	1,2	0,94	BBL: slightly thinned	Slight	19	19	14	17	1,7	1,21	BBL: slightly thinned	Slight
AS/2-AA	418	474	496	463	23,2	-			98	103	112	104	4,1	-			valid
DMSO									13	14	16	14	0,9				valid
A3847 Μutagenicity Experiment II - pre-incubation test - Strain S. typhimurium TA 1537
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	19	18	19	19	0,3	-	not evaluated	no cytotoxicity	18	19	17	18	0,6	-	not evaluated	no cytotoxicity	valid
0.0781	18	16	15	16	0,9	0,79	BBL: normal	no cytotoxicity	22	19	18	20	1,2	1,11	BBL: normal	no cytotoxicity	not mutagenic
0.156	16	20	18	18	1,6	0,95	BBL: normal	no cytotoxicity	17	18	21	19	1,2	1,06	BBL: normal	no cytotoxicity
0.313	20	21	20	20	0,3	1,05	BBL: normal	no cytotoxicity	19	18	17	18	0,6	1,00	BBL: normal	no cytotoxicity
0.625	23	21	20	21	0,9	1,05	BBL: normal	no cytotoxicity	20	19	24	21	1,5	1,17	BBL: normal	no cytotoxicity
1.25	18	20	22	20	1,2	1,16	BBL: normal	no cytotoxicity	23	19	23	22	1,3	1,22	BBL: normal	no cytotoxicity
2.50	19	23	23	22	1,3	1,53	BBL: slightly thinned	Slight	21	21	22	21	0,3	1,05	BBL: slightly thinned	Slight
9-AAc/2-AA	87	111	105	101	7,2				117	119	106	114	4				valid
DMSO	14	14	15	14	0,3	-			16	21	22	20	1,9	-			valid
A3847 Mutagenicity Experiment II - pre-incubation test - Strain E. coli WP2 uvrA
without metabolic activation									with metabolic activation								Conclusion
Test item (μL per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	30	31	27	29	1,2	-	not evaluated	no cytotoxicity	35	38	35	36	1	-	not evaluated	no cytotoxicity	valid
0.0625	26	27	27	27	0,3	1,00	BBL: normal	no cytotoxicity	31	31	31	31	0	0,86	BBL: normal	no cytotoxicity	not mutagenic
0.125	24	28	25	26	1,2	0,93	BBL: normal	no cytotoxicity	34	35	41	37	2,2	1,03	BBL: normal	no cytotoxicity
0.250	31	29	32	31	0,9	1,19	BBL: normal	no cytotoxicity	32	32	29	31	1	0,86	BBL: normal	no cytotoxicity
0.500	32	30	32	31	0,7	1,19	BBL: normal	no cytotoxicity	31	39	36	35	2,3	0,97	BBL: normal	no cytotoxicity
1.00	33	29	28	30	1,5	1,04	BBL: normal	no cytotoxicity	37	36	42	38	1,9	1,06	BBL: normal	no cytotoxicity
2.00	31	26	31	29	1,7	1,15	BBL: slightly thinned	Slight	31	32	39	34	2,5	0,94	BBL: slightly thinned	Slight
MMS/2-AA	184	163	151	166	9,6	5,59			206	219	194	206	7,2	6,44			valid
DMSO						-			28	35	34	32	2,2	-			valid

Applicant's summary and conclusion

Conclusions:

Not mutagenic in bacteria

Executive summary:

The mutagenicity potential in bacteria of Benzene sulphonic acid was assessed following official guideline OECD 471, Bacterial Reverse Mutation Test. The test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, for all the tested strains (TA98, TA 100, TA 1535, TA and Esc. Coli WP2) under the reported experimental