Sodium toluene-4-sulphonate

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:

2019

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 toluene-4-sulphonate

EC Number:

211-522-5

EC Name:

Sodium toluene-4-sulphonate

Cas Number:

657-84-1

Molecular formula:

C7H7O3S.Na

IUPAC Name:

4-methylbenzenesulfonic 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 (Toxicity 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: 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.

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

TheMain 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

Cytotoxicity - plate incorporation test without metabolic activation
Test item (μg per plate)	TA98	TA100	TA1535	TA1537	E.Coli wp2 UVRa	Bacteriotoxic effect	Cytotoxicity
Untreated	34	120	14	17	32	not evaluated	no cytotoxicity
50	35	124	19	26	27	bacterial background normal	no cytotoxicity
158	32	124	13	21	29	bacterial background normal	no cytotoxicity
500	30	128	12	18	31	bacterial background normal	no cytotoxicity
1580	32	130	15	17	26	bacterial background normal	no cytotoxicity
5000	31	120	13	17	30	bacterial background normal	no cytotoxicity
Rt/Rc 50	1,03	1,03	1,36	1,53	0,84
Rt/Rc 158	0,94	1,03	0,93	1,24	0,91
Rt/Rc 500	0,88	1,07	0,86	1,06	0,97
Rt/Rc 1580	0,94	1,08	1,07	1,00	0,81
Rt/Rc 5000	0,91	1,00	0,93	1,00	0,94
Cytotoxicity - plate incorporation test with metabolic activation
Test item (μg per plate)	TA98	TA100	TA1535	TA1537	E.Coli wp2 UVRa	Bacteriotoxic effect	Cytotoxicity
Untreated	31	116	20	22	38	not evaluated	no cytotoxicity
50	38	136	17	26	33	bacterial background normal	no cytotoxicity
158	37	134	15	24	34	bacterial background normal	no cytotoxicity
500	40	124	15	22	28	bacterial background normal	no cytotoxicity
1580	45	144	24	21	37	bacterial background normal	no cytotoxicity
5000	37	134	24	19	31	bacterial background normal	no cytotoxicity
Rt/Rc 50	1,23	1,17	0,85	1,18	0,87
Rt/Rc 158	1,19	1,16	0,75	1,09	0,89
Rt/Rc 500	1,29	1,07	0,75	1,00	0,74
Rt/Rc 1580	1,45	1,24	1,20	0,95	0,97
Rt/Rc 5000	1,19	1,16	1,20	0,86	0,82

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	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Cytotoxicity
Untreated	28	30	27	28	0,9	-		35	37	34	35	0,9	-		valid
313	28	31	25	28	1,7	1		31	31	33	32	0,7	0,91		not mutagenic
625	25	27	29	27	1,2	0,96		33	31	35	33	1,2	0,94
1250	26	30	29	28	1,2	1		34	31	36	34	1,5	0,97
2500	24	26	28	26	1,2	0,93		34	32	35	34	0,9	0,97
5000	29	24	29	27	1,7	0,96		38	38	34	37	1,3	1,06
2-Nitrofluorene/2-AA	190	196	202	196	3,5	7		431	456	510	466	23,3	13,71		valid
DMSO	28	26	29	28	0,9	-		33	35	35	34	0,7	0,97		valid
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	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Cytotoxicity
Untreated	132	126	132	130	2	-		143	144	140	142	1,2	-		valid
313	167	159	150	159	4,9	1,22		145	141	148	145	2	1,02		not mutagenic
625	171	166	168	168	1,5	1,29		153	149	149	150	1,3	1,06
1250	144	155	151	150	3,2	1,15		159	168	172	166	3,8	1,17
2500	163	166	169	166	1,7	1,28		174	178	173	175	1,5	1,23
5000	165	167	169	167	1,2	1,28		179	171	151	167	8,3	1,18
AS/2AA	647	719	724	697	24,9	5,36		1180	1008	1093	1094	49,7	7,76		valid
DMSO								138	139	146	141	2,5	0,99		valid
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	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Cytotoxicity
Untreated	14	13	14	14	0,3	-		15	14	13	14	0,6	-		valid
313	17	14	19	17	1,5	1,21		14	18	18	17	1,3	1,21		not mutagenic
625	18	14	14	15	1,3	1,07		19	18	16	18	0,9	1,29
1250	20	18	17	18	0,9	1,29		15	15	16	15	0,3	1,07
2500	14	18	16	16	1,2	1,14		19	15	18	17	1,2	1,21
5000	16	20	18	18	1,2	1,29		18	14	20	17	1,8	1,21
AS/2-AA	398	382	412	397	8,7	28,36		138	135	154	142	5,9	8,35		valid
DMSO								17	18	16	17	0,6	1,21		valid
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	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Cytotoxicity
Untreated	22	15	19	19	2	-		20	18	21	20	0,9	-		valid
313	21	16	22	20	1,9	1,18		19	19	18	19	0,3	0,95		not mutagenic
625	13	19	18	17	1,9	0,89		18	16	20	18	1,2	0,9
1250	20	20	18	19	0,7	1		25	18	18	20,3	2,3	1
2500	21	22	18	20	1,2	1,05		23	25	22	23	0,9	1,15
5000	21	20	22	21	0,6	1,11		18	22	19	20	1,2	1
9-AAc/2-AA	102	110	106	106	2,3	5,58		140	133	121	131	5,5	6,89		valid
DMSO	18	18	20	19	0,7	1,00		20	16	22	19	1,8	0,95		valid
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	Cytotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Cytotoxicity
Untreated	25	27	24	25	0,9	-		28	27	30	28	0,9	-		valid
313	24	24	25	24	0,3	0,96		35	35	34	35	0,3	1,25		not mutagenic
625	29	26	25	27	1,2	1,08		37	39	37	38	0,7	1,36
1250	26	25	27	26	0,6	1,04		36	40	42	39	1,8	1,11
2500	32	30	26	29	1,8	1,16		39	35	38	37	1,2	1,32
5000	30	30	26	29	1,3	1,16		42	36	33	37	2,6	1,32
MMS/2-AA	167	170	171	169	2	6,76		133	132	141	135,8	2,8	4,66		valid
DMSO								30	27	30	29	1	1,04		valid

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	Citotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Citotoxicity
Untreated	31	33	28	31	1,5	-		35	35	38	36	1	-		valid
313	30	34	30	31	1,3	1		31	39	39	36,7	2,7	1,00		not mutagenic
625	27	26	33	29	1,9	0,94		35	34	36	35	0,6	0,97
1250	30	31	34	32	1,2	1,03		35	35	35	35	0	0,97
2500	32	27	30	30	1,5	0,97		35	38	33	35	1,5	0,97
5000	27	34	30	30	2	0,97		37	38	34	36	1,2	1,00
2-Nitrofluorene/2-AA	171	196	183	183	7,2	5,38		618	674	592	628	24,2	17,44		valid
DMSO	33	35	34	34	0,6	-		34	36	39	36	1,5	1,00		valid
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	Citotoxicity	Revertants per plate			mean ± sd	s.e.	Rt/Rc	Citotoxicity
Untreated	144	153	139	145	4,1	-		135	163	151	150	8,1	-		valid
313	154	140	130	141	7	0,97		169	164	168	167	1,5	1,11		not mutagenic
625	149	152	154	152	1,5	1,05		175	176	170	174	1,9	1,16
1250	139	135	156	143	6,4	1,01		178	179	173	177,8	1,8	1,18
2500	139	141	144	141	1,5	0,97		166	148	151	155	5,6	1,03
5000	153	152	166	157	4,5	1,08		167	167	168	167,3	0,3	1,11
AS/2AA	718	776	841	778	35,5	5,37		1119	1387	1244	1250	77,4	9,26		valid
DMSO								130	131	145	135	4,8	0,90		valid
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	Citotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Citotoxicity
Untreated	14	16	13	14,9	0,9	-		18	15	15	16	1	-		valid
313	14	16	19	16	1,5	1,14		17	14	16	16,9	0,9	1,00		not mutagenic
625	12	14	15	14,9	0,9	1,00		13	13	15	14,7	0,7	0,88
1250	15	15	13	14,7	0,7	1,00		15	20	17	17	1,5	1,06
2500	13	14	16	14,9	0,9	1,00		18	15	16	16,9	0,9	1,00
5000	15	13	14	14	0,6	1,00		14	14	19	16,7	1,7	1,00
AS/2-AA	550	489	513	5177,7	17,7	36,93		121	122	111	118	3,5	7,38		valid
DMSO								15	14	19	16	1,5	1,00		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	Citotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Citotoxicity
Untreated	18	17	14	16	1,2	-		18	18	20	19,7	0,7	-		valid
313	18	20	21	20,9	0,9	1,25		20	22	18	20	1,2	1,05		not mutagenic
625	22	20	19	20,9	0,9	1,25		17	17	18	17,3	0,3	0,89
1250	17	17	17	17	0	1,06		18	19	17	18	0,6	0,95
2500	22	18	16	19,8	1,8	1,19		17	18	21	19	1,2	1,00
5000	16	15	18	16,9	0,9	1,00		19	15	18	17	1,2	0,89
9-AAc/2-AA	117	146	139	134	8,7	7,88		101	97	84	94	5,1	3,92		valid
DMSO	14	20	17	17	1,7	1,06		25	25	22	24	1	1,26		valid
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	Citotoxicity	Revertants per plate			mean	s.e.	Rt/Rc	Citotoxicity
water	24	28	26	26	1,2	-		38	30	31	33,5	2,5	-		valid
313	29	26	31	29	1,5	1,12		31	30	31	31,3	0,3	0,94		not mutagenic
625	23	22	28	24	1,9	0,92		31	27	29	29	1,2	0,88
1250	28	29	22	26,2	2,2	1,00		27	28	26	27	0,6	0,82
2500	28	25	26	26,9	0,9	1,00		39	35	30	35,6	2,6	1,06
5000	23	23	21	22,7	0,7	0,85		29	38	33	33	2,6	1,00
MMS/2-AA	184	197	163	181	9,9	6,96		226	198	243	222,33	13,1	7,67		valid
DMSO								29	27	32	29	1,5	0,88		valid

Applicant's summary and conclusion

Conclusions:

Nut mutagenic in bacteria

Executive summary:

The mutagenicity potential in bacteria of Sodium Toluene 4-sulphonate 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.