Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-[[4-(diethylamino)-6-[(4-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:4)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 08 to August 25, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

yes

Remarks:

deviation had no impact on the outcome of the study (details below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

deviation had no impact on the outcome of the study (details below)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-[diethylamino]-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Cas Number:

42355-78-2

Molecular formula:

C40H40N12Na4O12S4

IUPAC Name:

tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-[diethylamino]-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Method

Target gene:

his (trp) locus

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.

Metabolic activation:

with and without

Metabolic activation system:

S9 post mitochondrial fraction

Test concentrations with justification for top dose:

First experiment: 50, 150, 500, 1500 and 5000 µg per plate.
As neither precipitation nor toxicity was observed in the first experiments, the second experiments were done with the same dose-range. Experiments with metabolic activation differ from the first by different amount of S9 (30 µl – 5.7 %, 50 µl - 9.5 %, 100 µl – 19.0 %).

Vehicle / solvent:

Water for injection

Controls

Untreated negative controls:

yes

Remarks:

untreated controls contain no solvent

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml of H2O for injection

Positive controls:

yes

Remarks:

reference mutagens

Positive control substance:

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine (NPD), 2-aminofluorene (2-AF), 2-aminoanthracene (2-AA), 9-aminoacridine hydrochloride monohydrate (9-AAc)

Remarks:

Without S9: AS 1.5 µg/plate TA100 and TA1535; NPD 20 µg/plate TA98; 9 AAc 100 µg/plate TA1537; ENNG 20 µg/plate E. coli. With S9: 2-AA 1.0 µg/plate TA 1535, 2.5 µg/plate TA1537, 25 µg/plate E. coli; 2-AF 10 µg/plate TA100 and TA98.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, plate incorporation. 100 µl of test substance of required concentration, 100 µl of 16-18 h culture of tester strain, 0.5 ml relevant buffer and 30 µl of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 ml of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45 ± 3 °C. After shaking the mixture was poured into a minimal glucose agar plate.
Fresh solutions of test substance were prepared before each experiment. All concentrations of the test substance solution were dosed in the volume of 0.1 ml per plate.

PREPARATION and USING OF S9: the metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/ml, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 ml/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 °C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer. Each plate in all experiments with metabolic activation contained 0.5 ml of buffer with NADP and glucoso-6-phosphate and 30 µl S9 (the concentration of S9 in the S9mix was 5.7 %). In experiments without metabolic activation only buffer was added to the top agar.

DURATION
- Incubation period: 48 - 72 h at 37 ± 1 °C.

TOXICITY ASSAY: the starting solution (5000 µg/0.1 ml) was diluted to concentration series (10-5000 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation. No toxicity was observed in any dose.

COUNTER SYSTEM: after incubation the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.

NUMBER OF REPLICATIONS: triplicate plating was used at each dose level except in the toxicity test with strain TA 98, where test substance was tested in duplo. Each experiment was repeated.

GENOTYPES: genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as "biologically relevant":
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no evidence of a rising trend in the number of revertants with increasing dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no evidence of a rising trend in the number of revertants with increasing dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no evidence of a rising trend in the number of revertants with increasing dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no evidence of a rising trend in the number of revertants with increasing dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no evidence of a rising trend in the number of revertants with increasing dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Current historical ranges of revertant numbers in bacterial strains used in the study and live bacteria count used in the first experiments

Control	Spont.rev.	Water	PC -S9	PC +S9	Number of CFU/ml
S.t. TA 100	78 - 182	87 - 173	318 - 826	543 - 1571	1.06*10^9
S.t. TA 1535	10 - 30	7 - 30	257 - 625	68 - 260	7.87*10^8
S.t. TA 98	12 - 48	11 - 49	277 - 1557	533 - 2181	2.13*10^8
S.t. TA 1537	5 - 21	5 - 22	455 - 1399	59 - 279	7.43*10^8
E.c. WP2 uvrA	11 - 47	12 - 47	377 - 1097	0 - 387	9.40*10^8

Toxicity testing in strain TA 100

Test substance (mg per plate)	Revertants per plate				Rt/Rc	Bacteriotoxic effect
	Plates			x±sd
0	128	107	114	116 ± 9	-	not evaluated
water	117	99	112	109 ± 8	-	not evaluated
10	112	105	NT	109 ± 4	1.0	bacterial background normal
100	110	85	NT	98 ± 13	0.9	bacterial background normal
500	101	114	NT	108 ± 7	1.0	bacterial background normal
1000	137	135	NT	136 ± 1	1.2	bacterial background normal
2500	107	133	NT	120 ± 13	1.1	bacterial background normal
5000	113	111	NT	112 ± 1	1.0	bacterial background normal
AS	554	603	NT	579 ± 25	5.3	not evaluated

The results obtained in most experiments did not show substantial/biologically significant increases in the number of revertants versus solvent controls (Rt/Rc < 2) and no one of these experiments gave evidence of arising trend in the number of revertants with increasing dose.

Slight increase of number of revertants was observed in both experiments in TA 1537 with metabolic activation in the dose of 50 µg per plate where ratio of revertants against solvent control. Rt/Rc ratio reached 1.5 and 1.4, respectively. This increase is partially caused by lower solvent control compared with spontaneous reversion in both experiments. In the second experiment without metabolic activation slight increase is observed also in the dose of 1500 µg per plate. No such increase was observed in the first experiment. This result is not considered as biologically relevant (see criteria reported above).

Applicant's summary and conclusion

Conclusions:

The test substance was non-mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains tested without metabolic activation.

Executive summary:

Method

The test substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dissolved in dimethylsulfoxide and assayed in doses of (10) 50-5000 µg per plate, which were applied to plates in volume of 0.1 ml.

Experiments were performed without as well as with metabolic activation with rat liver and a mixture of cofactors.

Results

The test substance was non-mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains tested without metabolic activation.