Ammonium (xylenes and 4-ethylbenzene)sulfonates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: Read across from another member of the category

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:

2019

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:

Sodium (xylenes and 4-ethylbenzene) sulfonates

EC Number:

701-037-1

Molecular formula:

-

IUPAC Name:

Sodium (xylenes and 4-ethylbenzene) sulfonates

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 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: 5000, 1580, 500, 158 and 50 µg/plate
I and II Main assay: 5000, 2500, 1250, 625 and 313 µg/plate (no effect obsterved in the first main test and the same doses were used for the second main test)

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.

Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
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. Two 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 on each plate and evaluating the condition of the bacterial backgroundlawn. In the preliminary toxicity test, plates were held at approximately 4°C for 24 hours before scoring.

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.

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:

as per OECD 471 guideline

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

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. Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

Main assay: No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.
Neither toxicity, nor relevant increase in the number of revertant colonies was observed in the pre-incubation assay, at any dose level, with 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

A3689: Cytotoxicity - plate incorporation test without metabolic activation
Test item (μg per plate)	TA98	TA100	TA1535	TA1537	E.Coli wp2 UVRa	Bacteriotoxic effect	Cytotoxicity
Untreated	28	123	14	16	26	not evaluated	no cytotoxicity
50.0	31	120	17	22	23	BBL: normal	no cytotoxicity
158	31	110	16	19	27	BBL: normal	no cytotoxicity
500	35	121	16	16	24	BBL: normal	no cytotoxicity
1580	32	114	20	17	23	BBL: normal	no cytotoxicity
5000	26	122	17	19	31	BBL: normal	no cytotoxicity
Rt/Rc 50	1,11	0,98	1,21	1,38	0,88
Rt/Rc 158	1,11	0,89	1,14	1,19	1,04
Rt/Rc 500	1,25	0,98	1,14	1,00	0,92
Rt/Rc 1580	1,14	0,93	1,43	1,06	0,88
Rt/Rc 5000	0,93	0,99	1,21	1,19	1,19
A3689: Cytotoxicity - plate incorporation test with metabolic activation
Test item (μg per plate)	TA98	TA100	TA1535	TA1537	E.Coli wp2 UVRa	Bacteriotoxic effect	Cytotoxicity
Untreated	37	136	16	24	36	not evaluated	no cytotoxicity
50.0	35	114	14	18	39	BBL: normal	no cytotoxicity
158	38	120	15	22	37	BBL: normal	no cytotoxicity
500	33	115	14	22	35	BBL: normal	no cytotoxicity
1580	36	127	18	17	37	BBL: normal	no cytotoxicity
5000	33	123	16	21	40	BBL: normal	no cytotoxicity
Rt/Rc 50	0,95	0,84	0,88	0,75	1,08
Rt/Rc 158	1,03	0,88	0,94	0,92	1,03
Rt/Rc 500	0,89	0,85	0,88	0,92	0,97
Rt/Rc 1580	0,97	0,93	1,13	0,71	1,03
Rt/Rc 5000	0,89	0,90	1,00	0,88	1,11

A3689: Μutagenicity Experiment I - plate incorporation test - Strain: S. typhimurium TA 98
without metabolic activation									with metabolic activation								Conclusion
Test item (μg 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	27	27	32	29	1,7	-	not evaluated	no cytotoxicity	32	33	32	32	0,3	-	not evaluated	no cytotoxicity	valid
313	25	32	28	28	2,0	0,97	BBL: normal	no cytotoxicity	37	38	39	38	0,6	1,19	BBL: normal	no cytotoxicity	not mutagenic
625	30	26	30	29	1,3	1,00	BBL: normal	no cytotoxicity	32	39	41	37	2,7	1,16	BBL: normal	no cytotoxicity
1250	36	29	26	30	3	1,03	BBL: normal	no cytotoxicity	36	32	32	33	1,3	1,03	BBL: normal	no cytotoxicity
2500	36	29	27	31	2,7	1,07	BBL: normal	no cytotoxicity	31	33	36	33	1,5	1,03	BBL: normal	no cytotoxicity
5000	28	25	26	26	0,9	0,90	BBL: normal	no cytotoxicity	36	30	31	32	1,9	1,00	BBL: normal	no cytotoxicity
2-Nitrofluorene/2-AA	186	161	179	175	7,4	6,48			435	408	444	429	10,8	12,62			valid
DMSO	26	28	28	27	0,7	-			35	33	33	34	0,7	-			valid
A3689: Μutagenicity Experiment I - plate incorporation test - Strain: S. typhimurium TA 100
without metabolic activation									with metabolic activation								Conclusion
Test item (μg 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	124	119	128	124	2,6	-	not evaluated	no cytotoxicity	123	123	125	124	0,7	-	not evaluated	no cytotoxicity	valid
313	134	133	138	135	1,5	1,09	BBL: normal	no cytotoxicity	161	161	148	157	4,3	1,27	BBL: normal	no cytotoxicity	not mutagenic
625	138	132	124	131	4,1	1,06	BBL: normal	no cytotoxicity	146	153	163	154	4,9	1,24	BBL: normal	no cytotoxicity
1250	139	121	134	131	5,4	1,06	BBL: normal	no cytotoxicity	136	136	153	142	5,7	1,15	BBL: normal	no cytotoxicity
2500	116	121	111	116	2,9	0,94	BBL: normal	no cytotoxicity	118	142	113	124	9	1,00	BBL: normal	no cytotoxicity
5000	131	139	132	134	2,5	1,08	BBL: normal	no cytotoxicity	131	138	132	134	2,2	1,08	BBL: normal	no cytotoxicity
AS/2AA	571	553	529	551	12,2	4,44			1286	1008	1412	1235	119,3	10,12			valid
DMSO						-			124	123	119	122	1,5	-			valid
A3689: Μutagenicity Experiment I - plate incorporation test - Strain: S. typhimurium TA 1535
without metabolic activation									with metabolic activation								Conclusion
Test item (μg 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	13	16	14	14	0,9	-	not evaluated	no cytotoxicity	15	16	13	15	0,9	-	not evaluated	no cytotoxicity	valid
313	15	18	16	16	0,9	1,14	BBL: normal	no cytotoxicity	13	15	14	14	0,6	0,93	BBL: normal	no cytotoxicity	not mutagenic
625	12	18	14	15	1,8	1,07	BBL: normal	no cytotoxicity	13	14	17	15	1,2	1,00	BBL: normal	no cytotoxicity
1250	15	19	14	16	1,5	1,14	BBL: normal	no cytotoxicity	14	18	18	17	1,3	1,13	BBL: normal	no cytotoxicity
2500	18	15	19	17	1,2	1,21	BBL: normal	no cytotoxicity	16	14	18	16	1,2	1,07	BBL: normal	no cytotoxicity
5000	18	14	17	16	1,2	1,14	BBL: normal	no cytotoxicity	20	14	15	16	1,9	1,07	BBL: normal	no cytotoxicity
AS/2-AA	512	487	470	490	12,2	35,00			126	118	134	126	4,6	9,00			valid
DMSO						-			16	15	12	14	1,2	-			valid
A3689: Μutagenicity Experiment I - plate incorporation test - Strain: S. typhimurium TA 1537
without metabolic activation									with metabolic activation								Conclusion
Test item (μg 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	14	13	20	16	2,2	-	not evaluated	no cytotoxicity	22	21	24	22	0,9	-	not evaluated	no cytotoxicity	valid
313	20	19	19	19	0,3	1,19	BBL: normal	no cytotoxicity	18	18	16	17	0,7	0,77	BBL: normal	no cytotoxicity	not mutagenic
625	15	19	19	18	1,3	1,13	BBL: normal	no cytotoxicity	16	19	20	18	1,2	0,82	BBL: normal	no cytotoxicity
1250	15	17	20	17	1,5	1,06	BBL: normal	no cytotoxicity	16	14	17	16	0,9	0,73	BBL: normal	no cytotoxicity
2500	21	20	15	19	1,9	1,19	BBL: normal	no cytotoxicity	22	20	19	20	0,9	0,91	BBL: normal	no cytotoxicity
5000	14	15	18	16	1,2	1,00	BBL: normal	no cytotoxicity	22	17	20	20	1,5	0,91	BBL: normal	no cytotoxicity
9-AAc/2-AA	116	194	167	159	22,9	9,94			95	86	110	97	7	6,06			valid
DMSO	15	18	16	16	0,9	-			20	13	14	16	2,2	-			valid
A3689: Μutagenicity Experiment I - plate incorporation test - Strain: E. coli WP2 uvrA
without metabolic activation									without metabolic activation								Conclusion
Test item (μg 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	30	27	28	1,2	-	not evaluated	no cytotoxicity	27	30	24	27	1,7	-	not evaluated	no cytotoxicity	valid
313	25	31	33	30	2,4	1,07	BBL: normal	no cytotoxicity	29	35	35	33	2	1,22	BBL: normal	no cytotoxicity	not mutagenic
625	26	30	27	28	1,2	1,00	BBL: normal	no cytotoxicity	32	32	33	32	0,3	1,19	BBL: normal	no cytotoxicity
1250	24	30	24	26	2	0,93	BBL: normal	no cytotoxicity	31	32	35	33	1,2	1,22	BBL: normal	no cytotoxicity
2500	28	24	24	25	1,3	0,89	BBL: normal	no cytotoxicity	35	40	36	37	1,5	1,37	BBL: normal	no cytotoxicity
5000	26	26	30	27	1,3	0,96	BBL: normal	no cytotoxicity	39	32	38	36	2,2	1,33	BBL: normal	no cytotoxicity
MMS/2-AA	168	187	164	173	7,1	6,18			195	227	204	209	9,5	7,21			valid
DMSO						-			27	31	30	29	1,2	-			valid

A3689: Μutagenicity Experiment II - pre-incubation test - Strain: S. typhimurium TA 98
without metabolic activation									with metabolic activation								Conclusion
Test item (μg 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	32	30	32	31	0,7	-	not evaluated	no cytotoxicity	37	38	35	37	0,9	-	not evaluated	no cytotoxicity	valid
313	26	27	30	28	1,2	0,90	BBL: normal	no cytotoxicity	37	35	38	37	0,9	1,00	BBL: normal	no cytotoxicity	not mutagenic
625	28	29	32	30	1,2	0,97	BBL: normal	no cytotoxicity	36	39	27	34	3,6	0,92	BBL: normal	no cytotoxicity
1250	29	32	34	32	1,5	1,03	BBL: normal	no cytotoxicity	37	35	39	37	1,2	1,00	BBL: normal	no cytotoxicity
2500	25	28	33	29	2,3	0,94	BBL: normal	no cytotoxicity	39	39	36	38	1	1,03	BBL: normal	no cytotoxicity
5000	30	31	28	30	0,9	0,97	BBL: normal	no cytotoxicity	38	39	40	39	0,6	1,05	BBL: normal	no cytotoxicity
2-Nitrofluorene/2-AA	187	175	199	187	6,9	6,93			513	462	481	485	14,9	12,76			valid
DMSO	28	28	26	27	0,7	-			35	39	40	38	1,5	-			valid
A3689: Μutagenicity Experiment II - pre-incubation test - Strain: S. typhimurium TA 100
without metabolic activation									with metabolic activation								Conclusion
Test item (μg per plate)	Revertants per plate			mean	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity	Revertants per plate			mean ± sd	s.e.	Rt/Rc	Bacteriotoxic effect	Cytotoxicity
Untreated	145	153	161	153	4,6	-	not evaluated	no cytotoxicity	162	169	153	161	4,6	-	not evaluated	no cytotoxicity	valid
313	147	161	135	148	7,5	0,97	BBL: normal	no cytotoxicity	184	165	167	172	2	1,07	BBL: normal	no cytotoxicity	not mutagenic
625	158	173	142	158	9	1,03	BBL: normal	no cytotoxicity	171	154	158	161	5,1	1,00	BBL: normal	no cytotoxicity
1250	143	148	151	147	2,3	0,96	BBL: normal	no cytotoxicity	167	143	160	157	7,1	0,98	BBL: normal	no cytotoxicity
2500	162	175	160	166	4,7	1,08	BBL: normal	no cytotoxicity	172	166	153	164	5,6	1,02	BBL: normal	no cytotoxicity
5000	159	143	146	149	4,9	0,97	BBL: normal	no cytotoxicity	147	151	136	145	4,5	0,90	BBL: normal	no cytotoxicity
AS/2AA	712	664	559	645	45,2	4,22			1232	1128	1346	1235	63	7,87			valid
DMSO						-			147	158	167	157	5,8	-			valid
A3689: Μutagenicity Experiment II - pre-incubation test - Strain: S. typhimurium TA 1535
without metabolic activation									with metabolic activation								Conclusion
Test item (μg 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	13	13	15	14	0,7	-	not evaluated	no cytotoxicity	13	19	15	16	1,8	-	not evaluated	no cytotoxicity	valid
313	12	14	13	13	0,6	0,93	BBL: normal	no cytotoxicity	14	19	17	17	1,5	1,06	BBL: normal	no cytotoxicity	not mutagenic
625	18	19	16	18	0,9	1,29	BBL: normal	no cytotoxicity	18	23	18	20	1,7	1,25	BBL: normal	no cytotoxicity
1250	14	16	16	15	0,7	1,07	BBL: normal	no cytotoxicity	21	18	19	19	0,9	1,19	BBL: normal	no cytotoxicity
2500	13	16	14	14	0,9	1,00	BBL: normal	no cytotoxicity	21	22	17	20	1,5	1,25	BBL: normal	no cytotoxicity
5000	16	14	13	14	0,9	1,00	BBL: normal	no cytotoxicity	16	17	15	16	0,6	1,00	BBL: normal	no cytotoxicity
AS/2-AA	475	536	488	500	18,6	35,71			115	107	121	114	4,1	7,13			valid
DMSO									17	14	18	16	1,2	-			valid
Μutagenicity Experiment II - pre-incubation test - Strain: S. typhimurium TA 1537
without metabolic activation									with metabolic activation								Conclusion
Test item (μg 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	15	20	17	1,5	-	not evaluated	no cytotoxicity	18	18	19	18	0,3	-	not evaluated	no cytotoxicity	valid
313	15	18	22	18	2,0	1,06	BBL: normal	no cytotoxicity	18	17	16	17	0,6	0,94	BBL: normal	no cytotoxicity	not mutagenic
625	15	16	17	16	0,6	0,94	BBL: normal	no cytotoxicity	20	20	23	21	1	1,17	BBL: normal	no cytotoxicity
1250	17	19	15	17	1,2	1,00	BBL: normal	no cytotoxicity	19	19	20	19	0,3	1,06	BBL: normal	no cytotoxicity
2500	16	20	15	17	1,5	1,00	BBL: normal	no cytotoxicity	22	23	17	21	1,9	1,17	BBL: normal	no cytotoxicity
5000	22	14	18	18	2,3	1,06	BBL: normal	no cytotoxicity	24	24	25	24	0,3	1,33	BBL: normal	no cytotoxicity
9-AAc/2-AA	184	116	153	151	19,7	10,07			89	104	93	95	4,5	4,75			valid
DMSO	13	14	17	15	1,2	-			17	19	23	20	1,8	-			valid
A3689: Μutagenicity Experiment II - pre-incubation test- Strain: E. coli WP2 uvrA
without metabolic activation									without metabolic activation								Conclusion
Test item (μg 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	28	23	26	1,5	-	not evaluated	no cytotoxicity	28	29	31	29	0,9	-	not evaluated	no cytotoxicity	valid
313	24	23	22	23	0,6	0,88	BBL: normal	no cytotoxicity	33	32	37	34	1,5	1,17	BBL: normal	no cytotoxicity	not mutagenic
625	27	24	26	26	0,9	1,00	BBL: normal	no cytotoxicity	33	29	32	31	1,2	1,07	BBL: normal	no cytotoxicity
1250	29	29	33	30	1,3	1,15	BBL: normal	no cytotoxicity	32	32	32	32	0	1,10	BBL: normal	no cytotoxicity
2500	27	29	26	27	0,9	1,04	BBL: normal	no cytotoxicity	34	32	32	33	0,7	1,14	BBL: normal	no cytotoxicity
5000	27	23	24	25	1,2	0,96	BBL: normal	no cytotoxicity	33	29	35	32	1,8	1,10	BBL: normal	no cytotoxicity
MMS/2-AA	142	138	135	138	2	5,31			227	223	215	222	3,5	6,73			valid
DMSO									31	35	34	33	1,2	-			valid

Applicant's summary and conclusion

Conclusions:

Nut mutagenic in bacteria

Executive summary:

The mutagenicity potential in bacteria of Sodium (xylenes and 4-ethylbenzene) sulfonates 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 under the reported experimental conditions.