Tetrasodium 4,4'-bis[[4-[bis(2-hydroxypropyl)amino]-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-stilbene-2,2'-disulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 16 to July 14, 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted testing procedures and GLP procedures.

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:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 26th 1997

Deviations:

yes

Remarks:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 4,4'-bis[[4-[bis(2-hydroxypropyl)amino]-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-stilbene-2,2'-disulphonate

EC Number:

267-097-1

EC Name:

Tetrasodium 4,4'-bis[[4-[bis(2-hydroxypropyl)amino]-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-stilbene-2,2'-disulphonate

Cas Number:

67786-25-8

Molecular formula:

C44H48N12Na4O16S4

IUPAC Name:

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

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were kept at -196 ºC under liquid nitrogen. After activation, cells are grown in DMEM medium with L-glutamine and 10 % Fetal Bovine Serum in incubator (5 % CO2, 37 ± 1 °C, moistened). Cells underwent less then 12 passages after refreezing the original culture from cell collection before using for test.
Cleansing of cultures was performed 5-10 days before start of experiment (treatment) with complete medium supplemented with HAT supplement due to elimination of mutants.
- Periodically checked for Mycoplasma contamination: yes. Cell cultures were checked for mycoplasma contamination. At every experiment (3 hour treatment with metabolic activation, 3 hour treatment without metabolic activation, 24 hour treatment) one withdrawal of media was performed according to demand of testing laboratory for mycoplasma determination.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

5.0; 1.5; 0.,5 and 0.15 mg/ml.

Controls

Untreated negative controls:

yes

Remarks:

1 ml of DMEM and 9 ml of complete medium

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

9.9 or 9.95 ml of complete medium and 100 or 50 µl of relevant positive control diluted in DMSO. Without met. act.: EMS 5mM solution, 50 or 100 µl/plate. With met. act.: DMBAe, 5µg/mL– final concentration.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. The test substance was dissolved in the maximum concentration used in cytotoxicity test. 1 ml of the solution was dosed to 2 Petri dishes with nutrient broth and incubated for 72 hours at 37 ± 1 ºC. No contamination occurred at evaluation.

MEDIA
DMEM: minimal medium, part of complete growth medium
HAT suplement: cleansing medium for reduction of mutants in the start of experiment; 50x concentrated, for preparing of HAT medium
FBS: Fetal Bovine Serum, part of complete growth medium
Trypsin-EDTA (0.5 %) solution: for release of cells from the bottom of dishes
Atb: penicillin - (10000 U/mL) streptomycin (10000 µg/mL): for prevention of contamination, part of complete growth medium
Complete growth medium: DMEM:FBS:Atb = 949:50:1, prepared in laboratory

DYE for STAINING of COLONIES: Methylene blue, 0.1 % solution.

METABOLIC ACTIVATION
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 (a 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. 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.
Every lot of S9 was tested for sterility and activity in the Ames test with the aid of bacterial strain TA 98. Activity was within expected limits.
Cofactors (NADP and glucoso-6-phosphate) were dissolved in PBS. Each plate in all experiments with metabolic activation contained 4.9 mlof S9mix, 4 ml of complete medium and 1 ml of the test substance solution.

S9 mix preparation:
S9 tissue fraction: 3.0 ml
NADP (0.1 M): 0.4 ml
G-6-P (0.1 M): 0.5 ml
KC1 (0.33 M): 1.0 ml
MgCl2 (0.1 M): 0.5 ml
Phosphate Buffer (0.2 M): 4.6 ml
TOT: 10.0 ml

Plates for metabolic activation:
Complete medium: 4.0 ml
S9 mix: 5.0 ml
Control or test substance solution in DMEM: 1.0 ml
TOT: 10.0 ml

DURATION
In the first mutagenicity experiment, cells were treated for 3 hours (with as well as without metabolic activation; day 1).
In the second mutagenicity experiment, cells were treated for 24 hours (without metabolic activation only).

SELECTION AGENT: 6-thioguanine 98 %, 5 µg/ml; for selection of mutants.

NUMBER OF REPLICATIONS: every dose was tested in double. Citogenicity assey was performed in duble.

NUMBER OF CELLS EVALUATED: after treatment, approximately 10^6 cells were transferred to suitable number of dishes to seed enough cells with regard to toxicity. At the same time, cells were seeded for PE. 3rd and 5th day, approximately 10^6 cells from every culture were transferred and 8th and 10th day, extractions of mutants were performed with using selective medium together with PE estimation.

DETERMINATION OF CYTOTOXICITY
Treatment with the test substance resulted in slight toxicity at a maximum concentration of 5 mg/ml. No toxicity was observed in the other doses. On the basis of the result obtained, the concentration of 5 mg/ml was selected as the highest concentration to be used in the mutation assays.

DETERMINATION of SURVIVAL
After treatment period, the cultures were trypsinised and an aliquot (0.3 ml of 10^3/ml cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells.
A number of cells were then replaced in order to maintain the treated cell populations; the number of cells taken forward was adjusted according to the expected viability of the cultures, to give one million of viable cells. Cells were grown in 10 cm Petri dishes.

DETERMINATION of MUTANT FREQUENCY
At expression time (7 or 9 days), each culture was trypsinised, resuspended in complete medium and counted by microscopy. Then the following procedures were performed:
An adequate number of cells were sub cultured to maintain the treated populations of cells. This step is not performed on the 9th day.
After dilution, an estimated 100000 cells were plated in each of five 100 mm tissue culture Petri dishes. After 1 hour, 6-thioguanine was added to each the Petri dish. Only HGPRT mutant colonies are able to grow in the presence of 6-thioguanine; these plates were subsequently scored for the presence of mutants.
After dilution, an estimated 300 cells were plated in each of three 60 mm tissue culture Petri dishes. These plates were used to estimate plating efficiency.

ASSAY ACCEPTANCE CRITERIA
Negative Control: spontaneous mutant frequency should be 0-10 colonies per 10^5 planted cells.
Positive Control: positive control should fulfil the condition of positivity (at least two fold number of mutants against solvent control).
Plating efficiency (PE): PE of solvent control should be 50 % at least.

Evaluation criteria:

The main criterion for evaluation of results is modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). 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 mutations is at least twice as high as that in the solvent control for the strains having spontaneous mutation > 10;
- if the number of mutations 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

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the concentration of 5 mg/ml was selected as the highest concentration to be used in the mutation assays

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

CITOTOXICITY

Treatment with the test substance resulted in slight toxicity and survival was reduced to 47.0 % in the maximum dose without and as well as 62.1 % with metabolic activation. No toxicity was observed in the other doses.

On the basis of the result obtained, the concentration of 5 mg/ml was selected as the highest concentration to be used in the mutation assays. The lower concentrations were spaced approximately by a factor √10, in order to reach a concentration row.

According to these results it was decided to treat 2 Petri dishes in the first mutagenicity experiment in the dose of 5 mg/ml with as well as without metabolic activation. In the other doses single Petri dishes were treated.

For gaining of higher amount of treated cells, 2 Petri dishes were treated for every dose/control in the second mutagenicity experiment.

Toxicity test with metabolic activation

Dose (mg/ml)	Viability			avg	PE
	(number of colonies)
solvent	280	172	248	233.3	100
0.01	115	141	107	121.0	51.9
0.1	126	102	130	119.3	51.1
0.5	260	272	254	262.0	112.3
1.0	260	245	265	256.7	110
2.5	296	313	312	307.0	131.6
5.0	144	130	161	145.0	62.1
DMBA	123	80	109	104.0	44.6

Toxicity test without metabolic activation

Dose (mg/ml)	Viability			avg	PE
	(number of colonies)
solvent	284	248	300	277.3	100
0.01	184	215	201	200.0	72.1
0.1	240	232	248	240.0	86.5
0.5	253	223	287	254.3	91.7
1.0	205	232	213	216.7	78.1
2.5	217	228	241	228.7	82.5
5.0	137	138	116	130.3	47
EMS	398	303	347	349.3	126

PLATING EFFICIENCY

For the assessment of number of plated cells the PE was determined always after treatment. Both in the absence and presence of S9 metabolism the toxicity in mutagenicity experiments was less pronounced than it could be predicted from the results of the preliminary cytotoxicity test (survival about 68 % in experiment with and 84 % without metabolic activation).

No toxicity was observed in 24 hour experiment without metabolic activation what means, that all the planted cells were viable. If the test substance killed some cells, these cells disappeared during aspiration of media/trypsinisation process.

 

Plating efficiency after treatment and number of treated cells – experiment with metabolic activation (3 hours)

Dose	Viability			avg	PE	∑ NTC	NPC	∑ NPC
(mg/ml)	(number of colonies)
NC (1)	388	363	352	368	108.4	1.55E+06	1.29E+06
NC (2)	298	294	340	311	91.6	2.33E+06	1.09E+06	2.37E+06
0.15 (1)	322	268	296	295	87.1	1.07E+06	9.65E+05
0.15 (2)	306	312	321	313	92.3	9.90E+05	1.02E+06	1.99E+06
0.5 (1)	288	301	278	289	85.2	8.80E+05	8.19E+05
0.5 (2)	351	340	333	341	100.6	9.40E+05	1.02E+06	1.84E+06
1.5 (1)	304	302	286	297	87.7	1.09E+06	9.71E+05
1.5 (2)	286	271	288	282	83	1.47E+06	9.86E+05	1.96E+06
5.0 (1)	230	228	242	233	68.8	2.22E+06	1.24E+06
5.0 (2)	243	236	217	232	68.4	2.07E+06	1.24E+06	2.48E+06
DMBA (1)	373	339	309	340	100.3	2.67E+06	2.27E+06	2.27E+06
DMBA (2)	256	265	270	264	77.7	2.77E+06	1.76E+06	1.76E+06

Plating efficiency after treatment and number of treated cells – experiment without metabolic activation (3 hours, first mutagenicity experiment)

Dose	Viability			avg	PE	∑ NTC	NPC	∑ NPC
	(number of colonies)
NC (1)	301	284	296	294	107.6	1.24E+06	1.03E+06
NC (2)	241	256	260	252	92.4	1.32E+06	8.83E+05	1.91E+06
0.15 (1)	280	291	316	296	108.3	1.44E+06	1.03E+06
0.15 (2)	284	245	286	272	99.5	1.62E+06	9.51E+05	1.99E+06
0 .5 (1)	344	359	355	353	129.2	1.19E+06	1.23E+06
0.5 (2)	302	283	277	287	105.3	1.27E+06	1.01E+06	2.24E+06
1.5 (1)	247	260	249	252	92.3	1.62E+06	8.82E+05
1.5 (2)	347	344	287	326	119.4	8.33E+05	1.06E+06	1.95E+06
5.0 (1)	314	272	245	277	101.5	3.18E+06	1.29E+06
5.0 (2)	201	202	208	204	74.6	3.38E+06	9.50E+05	2.24E+06
EMS 50	291	254	226	257	94.1	3.31E+06	1.20E+06	1.20E+06
EMS 100	305	296	245	282	103.3	4.64E+06	1.65E+06	1.65E+06

Plating efficiency – after treatment and number of treated cells – experiment without metabolic activation (24 hours, second mutagenicity experiment)

Dose	Viability			avg	PE	∑ NTC	NPC	∑ NPC
	(number of colonies)
NC (1)	280	282	287	283	96.9	7.00E+06	1.32E+06
NC (2)	309	294	301	301	103.1	7.00E+06	1.41E+06	2.73E+06
0.15 (1)	276	274	300	283	97	5.27E+06	1.32E+06
0.15 (2)	328	303	327	319	109.3	4.00E+06	1.49E+06	2.81E+06
0.5 (1)	296	300	276	291	99.5	4.27E+06	1.36E+06
0.5 (2)	324	264	230	273	93.3	4.40E+06	1.27E+06	2.63E+06
1.5 (1)	279	273	295	282	96.6	4.47E+06	1.32E+06
1.5 (2)	293	324	323	313	107.2	3.10E+06	1.46E+06	2.78E+06
5.0 (1)	262	239	272	258	88.2	2.73E+06	1.37E+06
5.0 (2)	241	274	239	251	86	2.43E+06	1.34E+06	2.71E+06
EMS 50-3hours	231	218	232	227	77.7	2.93E+07	1.06E+06	1.06E+06
EMS 100	250	286	294	277	94.7	9.13E+06	1.29E+06	1.29E+06

MUTAGEBICITY

Summary of results: 3 hour treatment with metabolic activation

Dose (mg/ml)	Expression period 7 days		Expression period 9 days
	MF/10^5	Mt/Msc	MF/10^5	Mt/Msc
NC	1.83	1.00	1.52	1.00
0.15	3.15	1.72	1.61	1.05
0.5	1.47	0.80	1.32	0.87
1.5	0.90	0.49	0.67	0.44
5.0	1.35	0.73	1.69	1.11
DMBA	29.55	16.10	26.22	17.23
DMBA	18.91	10.31	16.65	10.94

Summary of results: 3 hour treatment without metabolic activation

Dose (mg/ml)	Expression period 7 days		Expression period 9 days
	MF/10^5	Mt/Msc	MF/10^5	Mt/Msc
NC	1.74	1.00	1.43	1.00
0.15	2.61	1.50	2.03	1.41
0.50	2.57	1.48	2.55	1.78
1.50	3.91	2.25	2.77	1.93
5.00	3.23	1.86	6.11	4.26
EMS50	18.00	10.36	15.09	10.56
EMS100	31.18	17.95	42.73	29.88

Summary of results: 24 hour treatment without metabolic activation

Dose (mg/ml)	Expression period 7 days		Expression period 9 days
	MF/10^5	Mt/Msc	MF/10^5	Mt/Msc
NC	1.98	1	2.62	1
0.15	4.02	2.03	2.82	1.08
0.50	4.45	2.25	3	1.14
1.50	4.05	2.05	2.67	1.02
5.00	5.91	2.99	3.93	1.5
EMS50	17.44	8.81	17.75	6.77
EMS100	33.02	16.68	27.49	10.5

Some results had to be excluded from evaluation-in two cases less than 150 of seeded cells grew in Petri dishes against expected 300. Three results were excluded because no colonies grew in PE dishes, so number of cell planted for mutant selection was unknown. These results are featured by cursive.

Mutation frequency of negative controls varied from 1.43 - 2.62 (average from two cases treated in one experiment) mutants per 10^5 planted cells.

Mutation frequency of positive control was sufficiently high and varied in intervals given as follows:

EMS 50 µlL: 15.09 – 18.00

EMS 100 µl: 27.49 - 42.73

DMBA: 16.65 - 29.55.

Mutation frequency after 3 hour treatment with metabolic activation was low in all doses.

Mutation frequency after 3 hour treatment without metabolic activation was increased in these cases:

dose 1.5 (1) mg/ml expression period 7 days 2.85 fold NC frequency;

dose 5.0 (1) mg/ml expression period 7 days 6.17 fold NC frequency;

dose 5.0 (2) mg/ml expression period 7 days 2.82 fold NC frequency;

and after 24 hour treatment:

dose 5.0 (1) mg/ml expression period 7 days 3.43 fold NC frequency.

No dose-response relationship was observed.

Two Mt/Msc were higher than 3, what can be consider as positive result. But in the case of 3 hour treatment, dose 5.0 (1) mg/ml expression period 7 days, where the highest value was reached, dishes with mutants were not normal-there were more small colonies and background was coloured too.

In the second case, result was rather caused by relatively small amount of planted cells, because it was not confirmed in any other result (the second 5.0 mg/ml-7 days, both 5.0 mg/ml-9 days.

Nevertheless, is possible to say, that the dose of 5.0 mg/ml slightly increased spontaneous level of mutants in experiments without metabolic activation.

However the conditions of positivity (given in chapter 3.9.) were not fulfilled in first mutagenicity experiment nor in the second mutagenicity experiment.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance was non-mutagenic for V79 cells with as well as without metabolic activation.

Executive summary:

Method

The test substance was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The performed test was based on EU method B.17. Mutagenicity – In Vitro Mammalian Cell Gene Mutation Test, which is analogous to the OECD Test Guideline No. 476. V79 hamster fibroblast were used for testing. The test substance was dissolved in DMEM and the test concentration were 0.15; 0.5; 1.5 and 5 mg/ml. Each dose was tested in two replicates.

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

A preliminary cytotoxicity assay (3-hour treatment) was performed at first. The test substance was assayed at a maximum recommended concentration of 5 mg/ml. Slight toxicity occurred in the maximum dose - 62 % of survival with metabolic activation and 47 % without metabolic activation.

First experiment (3 hour treatment) with the test substance followed then. Four doses were used derived from the maximum dose 5 mg/ml – 1.5; 0.5 and 0.15 mg/ml. Each concentration was tested in replicate was performed in double. The test was performed with as well as without metabolic activation.

Increased numbers of mutants were observed in some cases without dose-response dependence only in experiments without metabolic activation. No such increasing was observed in experiments with metabolic activation.

The second experiment was performed for clarification of the results of first experiment. The same doses were used and the treatment time of cells was extended to 24 hour.

Results

The second experiment gives no evidence of the mutagenicity of test substance.

The test substance was non-mutagenic for V79 cells without as well as with metabolic activation.