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Genetic toxicity: in vitro

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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 guidelineopen allclose all
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)
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

Constituent 1
Chemical structure
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/105 Mt/Msc MF/105 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/105 Mt/Msc MF/105 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/105 Mt/Msc MF/105 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.

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