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Toxicological information

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:
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 guidelineopen allclose all
Qualifier:
according to
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)
Qualifier:
according to
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
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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

MUTAGEBICITY

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 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/105 Mt/Msc MF/105 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/105 Mt/Msc MF/105 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.