Disodium 4,4'-bis[[4-anilino-6-[(2-hydroxyethyl)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:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From May 16 to July 14, 2014.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted testing procedures and GLP procedures. Justification for Read Across is detailed in the endpoint summary and in the Category Justification Report attached to the section 13.

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

as revised in 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.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

68971-49-3

Cas Number:

68971-49-3

IUPAC Name:

68971-49-3

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 °C 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.: DMBA, 5 µg/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 °C. 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 (4). 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. Composition of S9 mix was as follows:

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

Each plate in all experiments with metabolic activation contained 5 ml of S9mix, 4 ml of complete medium and 1 ml of the test substance solution.

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: concentration was tested in two replicates; cytogenicity assay was performed in triplicates.

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
The test substance was assayed at a maximum concentration of 5 mg/ml and five lower concentrations in space of 3 digit places. Treatment with the test substance resulted in almost no toxicity - survival was slightly reduced to 76 % in treatments 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.

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 subcultured 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. 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 76.3 % in the maximum concentration in experiment with metabolic activation. No toxicity was observed in the other concentrations.

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 concentration of 5 mg/ml with as well as without metabolic activation. In the other concentrations single Petri dishes were treated.

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

Toxicity test with metabolic activation

Conc.	Viability			avg	PE
(mg/ml)	(number of colonies)
solvent	280	172	248	233.3	100.0
0.01	200	232	230	220.7	94.6
0.10	206	240	267	237.7	101.9
0.50	192	201	190	194.3	83.3
1.00	267	273	244	261.3	112.0
2.50	203	223	214	213.3	91.4
5.00	193	157	184	178.0	76.3
DMBA	223	180	209	204.0	87.4

Toxicity test without metabolic activation

Conc. (mg/ml)	Viability			avg	PE
	(number of colonies)
solvent	284	248	300	277.3	100.0
0.01	345	306	285	312.0	112.5
0.10	233	233	231	232.2	83.8
0.50	234	250	238	240.7	86.8
1.00	276	270	260	268.7	96.9
2.50	266	267	246	259.7	93.6
5.00	277	263	245	261.7	94.4
EMS	398	303	347	349.3	126.0

PLATING EFFICIENCY

Cytotoxicity in 3 hour mutagenicity experiments was similar with as well as without metabolic activation – 82 and 79 % (average from duplicates) of surviving cells respectively. 

On the other hand toxicity about 55 % was observed in the highest concentration in 24 hour mutagenicity experiment without metabolic activation. The other concentrations were non-toxic.

 

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

Conc. (mg/ml)	Viability			avg	PE %	∑ NSC	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)	330	349	328	336	123	1.29E+06	1.17E+06
0.15 (2)	279	293	294	289	105.7	1.07E+06	9.72E+05	2.15E+06
0.5 (1)	244	274	281	266	97.6	1.42E+06	9.32E+05
0.5 (2)	302	283	277	287	105.3	1.13E+06	9.88E+05	1.92E+06
1.5 (1)	238	240	259	246	90.0	1.66E+06	8.60E+05
1.5 (2)	271	302	309	294	107.7	1.71E+06	1.03E+06	1.89E+06
5.0 (1)	217	235	193	215	78.8	1.93E+06	1.00E+06
5.0 (2)	222	221	208	217	79.5	2.53E+06	1.01E+06	2.02E+06
EMS 50	291	254	226	257	94.1	3.31E+06	1.20E+06
EMS100	305	296	245	282	103.3	4.64E+06	1.88E+06

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

Conc.(mg/mL)	Viability			avg	PE %	∑ NSC	NPC	∑ NPC
	(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)	288	295	269	284	83.7	1.93E+06	9.94E+05
0.15 (2)	281	245	267	264	77.9	1.19E+06	9.25E+05	1.92E+06
0.5 (1)	298	313	295	302	89.0	1.02E+06	9.87E+05
0.5 (2)	207	192	204	201	59.3	1.00E+06	6.57E+05	1.64E+06
1.5 (1)	273	275	297	282	83.0	1.07E+06	9.39E+05
1.5 (2)	299	301	326	309	91.0	9.89E+05	1.01E+06	1.95E+06
5.0 (1)	239	236	253	243	71.5	2.07E+06	1.13E+06
5.0 (2)	333	302	306	314	92.5	1.80E+06	1.46E+06	2.60E+06
DMBA	373	339	309	340	100.3	2.67E+06	2.27E+06
DMBA	256	265	270	264	77.7	2.78E+06	1.76E+06

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

Conc.(mg/ml)	Viability			avg	PE %	∑ NSC	NPC	∑ NPC
	(number of colonies)
NC (1)	280	282	287	283	96.9	7.07E+06	1.32E+06
NC (2)	309	294	301	301	103.1	7.07E+06	1.41E+06	2.73E+06
0.15 (1)	263	263	298	275	94.0	5.67E+06	1.28E+06
0.15 (2)	286	320	301	302	103.5	6.33E+06	1.41E+06	2.69E+06
0.5 (1)	300	268	292	287	98.1	5.13E+06	1.34E+06
0.5 (2)	320	322	338	327	111.8	6.13E+06	1.52E+06	2.86E+06
1.5 (1)	278	237	249	255	87.2	4.40E+06	1.19E+06
1.5 (2)	276	271	290	279	95.5	4.07E+06	1.30E+06	2.49E+06
5.0 (1)	161	161	150	157	53.9	2.43E+06	7.34E+05
5.0 (2)	141	150	148	146	50.1	2.83E+06	6.83E+05	1.42E+06
EMS 50-3hours	231	218	232	227	77.7	2.92E+07	1.06E+06
EMS 100-3 hours	250	286	294	277	94.7	9.13E+06	1.29E+06

MUTAGENICITY

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	4.90	2.82	3.16	2.21
0.5	2.77	1.60	3.82	2.67
1.5	2.75	1.58	1.44	1.00
5.0	2.66	1.53	4.40	3.07
DMBA	18.00	10.36	15.09	10.54
DMBA	31.18	17.95	42.73	23.29

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	1.39	1.00
0.15	0.91	0.50	0.78	0.56
0.50	2.04	1.11	0.59	0.42
1.50	3.56	1.94	1.83	1.32
5.00	1.59	0.87	1.69	1.22
EMS50	29.55	16.10	26.22	18.89
EMS100	18.91	10.31	16.65	12.00

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	1.00	2.62	1.00
0.15	1.38	0.70	2.05	0.78
0.50	7.14	3.61	3.38	1.26
1.50	7.42	3.75	3.69	1.41
5.00	3.30	1.66	3.31	1.26
EMS50	17.44	8.81	17.75	6.77
EMS100	33.02	16.68	34.37	13.10

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

Mutation frequency of negative controls varied from 1.43-2.62 (average from two duplicates treated in one experiment) mutants per 105 planted cells.

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

EMS 50 µl: 15.09 – 18.00

EMS 100 µl: 31.18 - 42.73

DMBA: 16.65 - 29.55.

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

Mutation frequency after 3 hour treatment without metabolic activation was increased above 3 fold MF of solvent control in following cases:

concentration 0.15 (1) mg/ml expression period 7 days 4.57 fold NC frequency*;

concentration 1.5 (1) mg/ml expression period 7 days 3.09 fold NC frequency*;

concentration 5.0 (1) mg/ml expression period 9 days 4.10 fold NC frequency;

and after 24 hour treatment:

concentration 0.5 (1) mg/ml expression period 7 days 5.79 fold NC frequency*;

concentration 1.5 (2) mg/ml expression period 7 days 3.75 fold NC frequency.

 

No dose-response relationship was observed.

In three of these cases low amount of cells was planted closely above 150 cells/plate against 300 cels planted (marked with asterisk*), what could partially explain these results. In the case of 0.15 and 1.5 mg/ml respectively positive result disappeared when two duplicates were evaluated together because of almost twofold amount of cells in the second duplicate.

After joining of duplicates, 3 positive results were observed, always in different concentrations and they were not confirmed by results obtained in the the second expression period.

Increased numbers of revertants appeared rather randomly; they were never confirmed by results of the second expression period and occurred in different concentrations in 3 and 24 hour 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 concentration 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. Nearly no toxicity occurred in any concentration. In the maximum concentration - 76 % of survival was observed in experiment with metabolic activation and 94 % without metabolic activation.

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

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

The second experiment was performed for clarification of the results of first experiment. The same concentrations were used and the treatment time of cells was extended to 24 hour. The second expreriment gives no evidence of the mutagenicity of test substance.

Results

The second expreriment 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.