4-hydroxybenzenesulphonic 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

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:

4-hydroxybenzenesulphonic acid

EC Number:

202-691-6

EC Name:

4-hydroxybenzenesulphonic acid

Cas Number:

98-67-9

Molecular formula:

C6H6O4S

IUPAC Name:

4-hydroxybenzenesulfonic acid

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:

Two batches 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 4009 (Toxiicity test) and 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:2.50, 1.25, 0.625, 0.313 and 0.156 μL/plate
TA1537 without metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA1537 with metabolic activation: 2.50, 1.25, 0.625, 0.313 and 0.156 μL/plate
TA100 with and wihtout metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA98 without metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA98 with metabolic activation: 2.50, 1.25, 0.625, 0.313 and 0.156 μL/plate
WP2 uvrA withouth metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
WP2 uvrA with metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 and 0.156 μL/plate

II Main assay:
TA1535 without metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA1535 with metabolic activation: 2.5, 1,25, 0,625, 0.313, 0.156 μL/plate
TA1537 without metabolic activation: 3.00, 1.50, 0.750, 0.375, 0.188, 0.0938 μL/plate
TA1537 with metabolic activation: 1.5, 0.750, 0.375, 0.188, 0.0938, 0.0469 μL/plate
TA100 with and wihtout metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA98 without metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
TA98 with metabolic activation: 2.50, 1.25, 0.625, 0.313 and 0.156 μL/plate
WP2 uvrA withouth metabolic activation: 5.00, 2.50, 1.25, 0.625, 0.313 μL/plate
WP2 uvrA with metabolic activation: 3.00, 1.50, 0.750, 0.375, 0.188, 0.0938 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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 and 1.58 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 and 1.58 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 and 1.58 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 µL/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 10

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

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, as indicated by thinning of the background lawn and/or reduction in revertant numbers was observed in the presence of S9 metabolic activation at the two highest dose levels with all tester strains with the exception of TA100 and WP2uvrA where toxic effects were noticed only at the highest concentration. In absence of S9 metabolism, toxicity was seen only at the highest dose level with TA1537, TA100 and WP2uvrA tester stains.Test item treatments did not induce any increase in revertant colonies with any tester
strain/activation condition combination

I Main assay: No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.
Moderate to slight toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers was observed at the highest or at the two highest dose levels both in the absence and presence of S9 metabolism, with all tester strains with the exception of TA1535 and TA98 tester strains in its absence.
II Main assay: Toxicity was observed at the highest dose level both in the absence and presence of S9 metabolism, with all tester strains with the exception of TA1535 and TA98 tester stains in its absence.
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

Cytotoxicity - plate incorporation test without metabolic activation
Test item (μL per plate)	TA98	TA100	TA1535	TA1537	E.Coli wp2 UVRa	Bacteriotoxic effect	Cytotoxicity
Untreated	34	120	14	17	32	not evaluated	no cytotoxicity
0.0500	30	114	15	17	26	BBL: normal	no cytotoxicity
0.158	31	118	20	20	24	BBL: normal	no cytotoxicity
0.500	33	112	22	22	32	BBL: normal	no cytotoxicity
1.58	28	108	17	18	31	BBL: normal	no cytotoxicity
5.00	31	110	18	10	24	BBL: slightly/moderately thinned (TA100, WP2 uvrA / TA1537)	Slight/moderate                      (TA100, WP2 uvrA / TA1537)
Rt/Rc 50	0,88	0,95	1,07	1,00	0,81
Rt/Rc 158	0,91	0,98	1,43	1,18	0,75
Rt/Rc 500	0,97	0,93	1,57	1,29	1,00
Rt/Rc 1580	0,82	0,90	1,21	1,06	0,97
Rt/Rc 5000	0,91	0,92	1,29	0,59	0,75
Cytotoxicity - plate incorporation test with metabolic activation
Test item (μL per plate)	TA98	TA100	TA1535	TA1537	E.Coli wp2 UVRa	Bacteriotoxic effect	Cytotoxicity
Untreated	31	116	20	22	38	not evaluated	no cytotoxicity
0.0500	34	115	17	24	35	BBL: normal	no cytotoxicity
0.158	41	106	22	22	35	BBL: normal	no cytotoxicity
0.500	36	107	16	14	36	BBL: normal	no cytotoxicity
1.58	31	105	16	21	31	BBL: slightly thinned (TA98, TA1535, TA1537)	Slight (TA98, TA1535, TA1537)
5.00	36	103	14	19	26	BBL: slightly/moderately thinned	Slight/moderate
Rt/Rc 50	1,10	0,99	0,85	1,09	0,92
Rt/Rc 158	1,32	0,91	1,10	1,00	0,92
Rt/Rc 500	1,16	0,92	0,80	0,64	0,95
Rt/Rc 1580	1,00	0,91	0,80	0,95	0,82
Rt/Rc 5000	1,16	0,89	0,70	0,86	0,68

A3690 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	30	30	31	30	0,3	-	not evaluated	no cytotoxicity	32	34	42	36	3,1	-	not evaluated	no cytotoxicity	valid
0.156				Not Tested					28	32	33	31	1,5	0,86	BBL: normal	no cytotoxicity	not mutagenic
0.313	26	29	24	26	1,5	0,87	BBL: normal	no cytotoxicity	27	34	38	33	3,2	0,92	BBL: normal	no cytotoxicity
0.625	25	24	26	25	0,6	0,83	BBL: normal	no cytotoxicity	36	33	32	34	1,2	0,94	BBL: normal	no cytotoxicity
1.25	29	28	25	27	1,2	0,90	BBL: normal	no cytotoxicity	40	36	36	37	1,3	1,03	BBL: normal	no cytotoxicity
2.50	22	28	26	25	1,8	0,83	BBL: normal	no cytotoxicity	32	36	35	34	1,2	0,94	BBL: slightly thinned	slight
5.00	25	27	29	27	1,2	0,90	BBL: normal	no cytotoxicity				Not Tested
2-Nitrofluorene/2-AA	153	167	196	172	12,7	6,37			514	562	496	524	19,7	15,41			valid
DMSO	27	27	26	27	0,3	-			32	36	34	34	1,2	-			valid
A3690 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	119	109	109	112	3,3	-	not evaluated	no cytotoxicity	126	118	121	122	2,3	-	not evaluated	no cytotoxicity	valid
0.313	113	112	121	115	2,8	1,03	BBL: normal	no cytotoxicity	132	127	122	127	2,9	1,04	BBL: normal	no cytotoxicity	not mutagenic
0.625	109	114	120	114	3,2	1,02	BBL: normal	no cytotoxicity	111	112	109	111	0,9	0,91	BBL: normal	no cytotoxicity
1.25	110	121	118	116	3,3	1,04	BBL: normal	no cytotoxicity	123	124	117	121	2,2	0,99	BBL: normal	no cytotoxicity
2.50	124	128	127	126	1,2	1,13	BBL: normal	no cytotoxicity	136	121	127	128	4,4	1,05	BBL: normal	no cytotoxicity
5.00	117	110	108	112	2,7	1,00	BBL: slightly thinned	slight	108	112	117	112	2,6	0,92	BBL: slightly thinned	slight
AS/2AA	670	582	616	623	25,6	5,56			1202	1180	1270	1217	27,1	10,23			valid
DMSO						-			118	116	123	119	2,1	-			valid
A3690 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	15	13	13	14	0,7	-	not evaluated	no cytotoxicity	13	18	15	15	1,5	-	not evaluated	no cytotoxicity	valid
0.156				Not Tested					19	16	18	18	0,9	1,20	BBL: normal	no cytotoxicity	not mutagenic
0.313	17	13	14	15	1,2	1,07	BBL: normal	no cytotoxicity	14	15	17	15	0,9	1,00	BBL: normal	no cytotoxicity
0.625	22	15	19	19	2,0	1,36	BBL: normal	no cytotoxicity	19	17	15	17	1,2	1,13	BBL: normal	no cytotoxicity
1.25	14	15	16	15	0,6	1,07	BBL: normal	no cytotoxicity	13	13	16	14	1	0,93	BBL: normal	no cytotoxicity
2.50	15	15	16	15	0,3	1,07	BBL: normal	no cytotoxicity	18	14	14	15	1,3	1,00	BBL: slightly thinned	slight
5.00	14	15	14	14	0,3	1,00	BBL: normal	no cytotoxicity				Not Tested
AS/2-AA	422	474	396	431	22,9	30,79			178	182	190	183	3,5	11,44			valid
DMSO						-			18	16	13	16	1,5	-			valid
A3690 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	17	14	20	17	1,7	-	not evaluated	no cytotoxicity	21	22	25	23	1,2	-	not evaluated	no cytotoxicity	valid
0.156	19	18	18	18	0,3	1,06	BBL: normal	no cytotoxicity	18	17	16	17	0,6	0,74	BBL: normal	no cytotoxicity	not mutagenic
0.313	14	12	17	14	1,5	0,82	BBL: normal	no cytotoxicity	17	14	22	18	2,3	0,78	BBL: normal	no cytotoxicity
0.625	14	13	12	13	0,6	0,76	BBL: normal	no cytotoxicity	23	23	19	22	1,3	0,96	BBL: normal	no cytotoxicity
1.25	19	18	18	18	0,3	1,06	BBL: normal	no cytotoxicity	15	17	17	16	0,7	0,70	BBL: slightly thinned	slight
2.50	16	13	13	14	1,0	0,82	BBL: slightly thinned	slight	16	19	15	17	1,2	0,74	BBL: moderately thinned	moderate
5.00	10	10	11	10	0,3	0,59	BBL: moderately thinned / RRN	moderate				Not Tested
9-AAc/2-AA	128	99	104	110	9	6,88			137	142	126	135	4,7	6,75			valid
DMSO	14	16	18	16	1,2	-			20	18	22	20	1,2	-			valid
A3690 Μutagenicity 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	26	25	25	25	0,3	-	not evaluated	no cytotoxicity	33	37	40	37	2,0	-	not evaluated	no cytotoxicity	valid
0.156				Not Tested					29	35	35	33	2,0	0,89	BBL: normal	no cytotoxicity	not mutagenic
0.313	26	29	33	29	2	1,16	BBL: normal	no cytotoxicity	30	31	33	31	0,9	0,84	BBL: normal	no cytotoxicity
0.625	27	28	27	27	0,3	1,08	BBL: normal	no cytotoxicity	33	36	37	35	1,2	0,95	BBL: normal	no cytotoxicity
1.25	26	26	31	28	1,7	1,12	BBL: normal	no cytotoxicity	38	38	39	38	0,3	1,03	BBL: normal	no cytotoxicity
2.50	30	34	26	30	2,3	1,20	BBL: normal	no cytotoxicity	36	39	41	39	1,5	1,05	BBL: slightly thinned	slight
5.00	26	24	31	27	2,1	1,08	BBL: slightly thinned	slight	36	39	39	38	1,0	1,03	BBL: moderately thinned	moderate
MMS/2-AA	149	163	151	154	4,4	6,16			217	202	233	217	9	5,86			valid
DMSO						-			35	40	36	37	1,5	-			valid

A3690 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	Citotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Citotoxicity
Untreated	31	34	29	31	1,5	-	not evaluated	no cytotoxicity	34	33	34	34	0,3	-	not evaluated	no cytotoxicity	valid
0.156				Not Tested					38	32	35	35	1,7	1,03	BBL: normal	no cytotoxicity	not mutagenic
0.313	25	25	31	27	2,0	0,87	BBL: normal	no cytotoxicity	42	43	41	42	0,6	1,24	BBL: normal	no cytotoxicity
0.625	26	24	28	26	1,2	0,84	BBL: normal	no cytotoxicity	38	39	36	38	0,9	1,12	BBL: normal	no cytotoxicity
1.25	35	32	28	32	2,0	1,03	BBL: normal	no cytotoxicity	35	38	33	35	1,5	1,03	BBL: normal	no cytotoxicity
2.50	27	34	30	30	2,0	0,97	BBL: normal	no cytotoxicity	32	33	36	34	1,2	1,00	BBL: slightly thinned	slight
5.00	33	27	34	31	2,2	1,00	BBL: normal	no cytotoxicity				Not Tested
2-Nitrofluorene/2-AA	186	174	203	188	8,4	6,71			453	566	594	538	43,1	14,94			valid
DMSO	28	30	25	28	1,5	-			37	39	33	36	1,8	-			valid
A3690 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	Citotoxicity	Revertants per plate			mean ± sd	s.e.	Rt/Rc	Bacteriotoxic effect	Citotoxicity
Untreated	121	115	116	117	1,9	-	not evaluated	no cytotoxicity	136	131	131	133	1,7	-	not evaluated	no cytotoxicity	valid
0.313	138	143	119	133	7,3	1,14	BBL: normal	no cytotoxicity	148	155	159	154	3,2	1,16	BBL: normal	no cytotoxicity	not mutagenic
0.625	128	127	145	133	5,8	1,14	BBL: normal	no cytotoxicity	160	168	171	166	3,3	1,25	BBL: normal	no cytotoxicity
1.25	156	143	137	145	5,6	1,24	BBL: normal	no cytotoxicity	146	154	163	154	4,9	1,16	BBL: normal	no cytotoxicity
2.50	151	144	148	148	2,0	1,26	BBL: normal	no cytotoxicity	172	173	168	171	1,5	1,29	BBL: normal	no cytotoxicity
5.00	138	128	126	131	3,7	1,12	BBL: slightly thinned	slight	142	150	161	151	5,5	1,14	BBL: slightly thinned	slight
AS/2AA	582	618	771	657	57,9	5,62			1384	1021	1132	1179	107,4	9,07			valid
DMSO						-			125	136	128	130	3,3	-			valid
A3690 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	Citotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Citotoxicity
Untreated	14	17	14	15	1	-	not evaluated	no cytotoxicity	19	14	14	16	1,7	-	not evaluated	no cytotoxicity	valid
0.156				Not Tested					18	15	18	17	1	1,06	BBL: normal	no cytotoxicity	not mutagenic
0.313	17	13	13	14	1,3	0,93	BBL: normal	no cytotoxicity	16	13	18	16	1,5	1,00	BBL: normal	no cytotoxicity
0.625	13	16	16	15	10	1,00	BBL: normal	no cytotoxicity	15	15	17	16	0,7	1,00	BBL: normal	no cytotoxicity
1.25	15	18	19	17	1,2	1,13	BBL: normal	no cytotoxicity	19	16	13	16	1,7	1,00	BBL: normal	no cytotoxicity
2.50	18	20	16	18	1,2	1,20	BBL: normal	no cytotoxicity	18	15	13	15	1,5	0,94	BBL: slightly thinned	slight
5.00	19	18	13	17	1,9	1,13	BBL: normal	no cytotoxicity				Not Tested
AS/2-AA	458	532	506	499	21,7	33,27			103	110	96	103	4,0	6,87			valid
DMSO									16	13	16	15	1,0	-			valid
A3690 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	Citotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Citotoxicity
Untreated	21	20	22	21	0,6	-	not evaluated	no cytotoxicity	23	22	19	21	1,2	-	not evaluated	no cytotoxicity	valid
0.0469				Not Tested					23	28	23	25	1,7	1,19	BBL: normal	no cytotoxicity	not mutagenic
0.0938	23	22	22	22	0,3	1,05	BBL: normal	no cytotoxicity	21	18	21	20	1	0,95	BBL: normal	no cytotoxicity
0.0188	21	19	21	20	0,7	0,95	BBL: normal	no cytotoxicity	21	18	24	21	1,7	1,00	BBL: normal	no cytotoxicity
0.375	19	25	21	22	1,8	1,05	BBL: normal	no cytotoxicity	22	20	28	23	2,4	1,10	BBL: normal	no cytotoxicity
0.75	19	24	19	21	1,7	1,00	BBL: normal	no cytotoxicity	20	19	26	22	2,2	1,05	BBL: normal	no cytotoxicity
1.5	19	20	21	20	0,6	0,95	BBL: normal	no cytotoxicity	14	15	17	15	0,9	0,71	BBL: slightly thinned	slight
3.00	20	20	24	21	1,3	1,00	BBL: slightly thinned	slight				Not Tested
9-AAc/2-AA	95	164	112	124	20,8	6,20			88	96	101	95	3,8	5,00			valid
DMSO	20	20	19	20	0,3	-			18	19	21	19	0,9	-			valid
A3690 Μutagenicity 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	Citotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Citotoxicity
Untreated	23	22	26	24	1,2	-	not evaluated	no cytotoxicity	30	34	29	31	1,5	-	not evaluated	no cytotoxicity	valid
0.0938				Not Tested without S9					27	31	24	27	2,0	0,87	BBL: normal	no cytotoxicity	not mutagenic
0.0188				Not Tested without S9					32	32	28	31	1,3	1,00	BBL: normal	no cytotoxicity
0.313	23	26	24	24	0,9	1,00	BBL: normal	no cytotoxicity				Not Tested with S9
0.375				Not Tested without S9					26	29	33	29	2,0	0,94	BBL: normal	no cytotoxicity
0.625	29	21	28	26	2,5	1,08	BBL: normal	no cytotoxicity				Not Tested with S9
0.75				Not Tested without S9					32	26	30	29	1,8	0,94	BBL: normal	no cytotoxicity
1.25	27	22	24	24	1,5	1,00	BBL: normal	no cytotoxicity				Not Tested with S9
1.5				Not Tested without S9					31	27	32	30	1,5	0,97	BBL: normal	no cytotoxicity
2.5	34	29	25	29	2,6	1,21	BBL: normal	no cytotoxicity				Not Tested with S9			BBL: normal	no cytotoxicity
3.00				Not Tested without S9					29	30	34	31	1,5	1,00	BBL: slightly thinned	slight
5.00	31	35	29	32	1,8	1,33	BBL: slightly thinned	slight				Not Tested with S9
MMS/2-AA	163	142	151	152	6,1	6,33			212	176	148	179	18,5	6,39			valid
DMSO									30	27	27	28	1,0	-			valid

Applicant's summary and conclusion

Conclusions:

Nut mutagenic in bacteria

Executive summary:

The mutagenicity potential in bacteria of Hydroxybenzene 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 conditions.