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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:
HPRT method
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2015
Deviations:
yes
Remarks:
detailed below
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)
Type of assay:
other: gene mutation in mammalian cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: frozen permanent cell culture from European Collection of cells cultures; cells kept at -196 °C under liquid nitrogen. After activation, cells were grown in Dulbecco's modified Eagles's medium with 4 mM L-glutamine and 10 % FBS in humidified incubator (5 % CO2, 37±1 °C).
Cells underwent maximum 5 passages after thawing the original culture delivered
from cell collection before using for mutagenicity testing.
Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. Cleansing was not performed in experiment without metabolic activation, because of bad growth of cells in HAT medium.

MEDIA USED
- DMEM (Dulbecco's minimal essential medium): minimal medium, part of complete growth medium
- HAT supplement: liquid mixture of sodium hypoxanthine (5 mM), aminopterin (20 µM) and thymidine (0.8 mM). HAT-supplemented medium is suitable for post-fusion selection against unfused or self-fused HGPRT–myeloma cells. Hypoxanthine and thymidine supply preformed purines and pyrimidines for DNA synthesis by hybridomas via the salvage pathway that utilises HGPRT- contributed by the fused spleen cell. Aminopterin, a folic acid antagonist, inhibits the de novo nucleoside biosynthesis pathway. For HAT medium is diluted 50×. It is used as cleansing medium for reduction of mutants at the start of experiment
- 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 = 500 : 55 : 5.5, prepared in laboratory.

- Periodically checked for mycoplasma contamination: yes
Additional strain / cell type characteristics:
other: HPRT deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and a mixture of cofactors
Test concentrations with justification for top dose:
Test substance is soluble in water till 25.22 g/l. Test substance was dissolved in assay medium (DMEM). The highest recommended concentration is 2 mg/ml.
On the basis of cytotoxicity test results, the mutagenicity test with metabolic activation was arranged with range of concentrations 0.02-0.2 mg/ml. Concentrations for the mutagenicity test without metabolic activation were 0.01-0.04 mg/ml.
Vehicle / solvent:
- Solvent used: DMEM
Controls
Untreated negative controls:
yes
Remarks:
DMEN
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Rinsing of cells after treatment: PBS prepared in laboratory
Selective agent: 6-thioguanine 98 % diluted in 0.5 % Na2CO3, 5 µg/ml (final concentration) for selection of mutants
Dye for staining of colonies: methylene blue, 0.1 % solution

Cytotoxicity
The first cytotoxicity experiment was performed with as well as without metabolic activation. The concentrations for cytotoxicity were in range 0.01-2.0 mg/ml.
The first experiment failed in determination of doses for the mutagenicity tests, because doses with low, medium and high cytotoxicity were not possible to determine. Toxicity test had to be repeated for two more times.
On the basis of cytotoxicity test results, the mutagenicity test with metabolic activation was arranged with range of concentrations 0.02-0.2 mg/ml. Concentrations for the mutagenicity test without metabolic activation were 0.01-0.04 mg/ml.

Treatment: at least 2E+06 cells will be treated for each concentration/control for 3 hours. After treatment, every dish will be trypsinised and 300 cells will be seeded to 3 Petri dishes. Relative viability will be counted by comparison of viability in single concentration to viability in solvent control.

Mutation assay procedure
Each concentration was tested in two simultaneously performed independent runs (duplicates).
Experimental design
Concentrations for mutagenicity experiments were chosen on the basis of cytotoxicity testing. No precipitation was observed in any concentration in experiment with as well as without metabolic activation.
Test substance was dosed to dishes with cells in volume of 100 μl.
Cells were treated for 3 hours (with as well as without metabolic activation; day 1). After treatment, approximately 2E+06 cells were transferred to suitable number of dishes to seed enough cells. At the same time, cells were seeded for detection of number of cells (PE estimation).
On the 3rd, 6th and 8th day, approximately 2E+06 cells from every culture were transferred and 10th day, extractions of mutants was performed with using selective medium together with PE estimation again.

Experimental design (3 hours treatment)
Day Activity
1 treatment, passage of 1E+06 cells, plating of an aliquot (300 cells) for estimating of viability
3 passage of approx. 2E+06 cells
6 passage of approx. 2E+06 cells
8 passage of approx. 2E+06 cells
10 extraction of mutant cells, plating of an aliquot (300 cells) for estimating of viability

Fresh solutions of test substance were prepared for every experiment; solutions were prepared on a weight/volume in volumetric vials.
Neither assay of test substance stability, nor assays of its concentration and homogeneity in vehicle was undertaken.

Determination of survival
After treatment period, the cultures were trypsinised and an aliquot (0.3 ml of 1000 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 two millions of viable cells. Cells were grown in 10 cm Petri dishes.

Subculturing
On day 3, 6 and 8, cell populations were subcultured in order to maintain them in exponential growth. The number of cells taken forward was adjusted according to the expected viability, to give two millions viable cells seeded in 10 cm Petri dishes.

Incubation, staining and scoring
Survival and plating efficiency plates were incubated for at least six days (37±1 ºC, 5 % CO2, moistened) prior to scoring. Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days). After incubation, the plates were stained with methylene blue and colonies were scored.

Determination of mutant frequency
At expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. Then, an adequate number of cells were subcultured to maintain the treated populations of cells. This step is not performed on day 10.
After dilution, an estimated 220000 cells were plated in each of ten 100 mm tissue culture Petri dishes (together 2200000 cells). After about 1 hour, 6-thioguanine was added to each Petri dish to final concentration of 5 µg/ml. Only HPRT 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.
Evaluation criteria:
Each experiment will be evaluated separately using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987.
The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive than a repeated experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions [i.e. S9 concentration or S9 origin]) could be performed.

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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility
Precipitation occurred in the first toxicity test starting from 1.0 mg/ml. In the end, due to cytotoxicity of test substance, lower concentrations were used for mutagenicity testing in which no precipitation occurred.

Cytotoxicity experiments
In the first cytotoxicity test (concentration range 0.01 - 2 mg/ml) with as well as without metabolic activation, cytotoxicity was seen at concentration of 0.01 (without metabolic activation) and 0.1 (with metabolic activation). A second experiment was necessary to be performed for determination of doses with high, medium and low cytotoxicity.

Concentrations between 0.0005 - 0.01 mg/ml were used for second cytotoxicity experiment without metabolic activation. Concentrations 0.0025 - 0.05 mg/ml were used for the second cytotoxicity experiment with metabolic activation.
In this experiment doses appropriate for mutagenicity testing were not found because of no or small toxicity.
The third experiment had to be done for clarification. Concentrations 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 and 0.5 were used without metabolic activation; concentrations 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.50 and 0.75 mg/ml were used with metabolic activation.
Based on all the cytotoxicity tests, the following concentrations were determined for mutagenicity testing:
- without metabolic activation: 0.01; 0.015; 0.02; 0.025, 0.030 and 0.040 mg/ml
- with metabolic activation: 0.02, 0.05, 0.1 and 0.2 mg/ml.

Cytotoxicity in mutagenicity experiments
Experiment without metabolic activation was started with 6 concentrations, with the idea to choose the 4 best concentrations (OECD 476).
Cytotoxicity immediately after treatment and after the first plating for cultivation to allow expression of the mutant phenotype was recorded. During further passages, influenced cells were either growing slower than control or died. Overall, decrease of cells in all of the three passages was seen.
Concentration of 0.02 mg/ml was similarly toxic as the surrounding concentrations 0.015 and 0.025 mg/ml; so it was not kept untill the end of experiment.

Experiment with metabolic activation was started at concentrations of 0.02, 0.05, 0.1 and 0.2 mg/ml; concentration of 0.4 mg/ml was added during the mutagenicity because no toxicity was observed immediately after treatment in the lower concentrations.
Concentrations of 0.2 and 0.4 mg/ml were completely cytotoxic after plating for other growing so they were not continued. Toxicity of concentration 0.1 mg/ml was in accordance with OECD 476 but less than 2 millions of cells were transferred for other growing.
Additional experiment was performed with concentrations 0.075 and 0.1 mg/ml to get another suitable concentration for evaluation. In the end, four concentrations were available for 0.02, 0.05, 0.075, 0.1 mg/ml.

pH determination
conc. mg/ml measured pH
DMEM 7.62
0.02 7.64
0.05 7.65
0.1 7.65
0.2 7.65

Addition of test substance to cultivation medium changed pH of treatment solutions by 0.03. No pH adjustment was needed.

Mycoplasma determination
Three withdrawals of medium were performed: experiment without metabolic activation, experiment with metabolic activation and additional experiment with metabolic activation, after minimum of 14 days of growing of cells.
Result of test sample was negative so all media after cultivation of cells were free of mycoplasma.

Plating efficiency
For the assessment of number of plated cells, PE was determined always after treatment.
In all concentrations used in all experiments more than 2 millions cells were influenced in single replicates. In both experiments with metabolic activation and concentration of 0.1 mg/ml less than 2 millions of cells were seeded for further cultivation. In the additional experiment in both replicates together, condition of 2 millions cells was fulfilled.

Mutagenicity
Mutation frequency of cells treated with test substance in both experiments was low in all concentrations; Mt/Msc ratios generally never exceeded 3 fold limits (in fact all of them were below 2 fold vs solvent control). At the same time, no dose dependence was observed in any experiment.

Assay acceptance criteria compliance
Mutation frequencies of negative control (DMEM) were 1.07-2.12 mutants per 1E+05 plated cells. Historical control range (97.5 % confidence interval) is 0.78-2.28 mutants per 1E+05cells (123 entries).
Mutation frequency of positive controls was sufficiently high:
EMS 50 µl: 18.97; historical control range (95 % confidence interval) is 10.07 – 18.22 mutants per 1E+05cells (40 entries);
EMS 100 µl: 44.14; historical control range (95 % confidence interval) is 19.23 – 37.41 mutants per 1E+05cells (41 entries);
DMBA: 30.69-38.06 what is an evidence of good function of test system; historical control range (95 % confidence interval) is 11.52 – 35.12 mutants per 1E+05cells (54 entries).





Any other information on results incl. tables

Mutagenicity without metabolic activation, 3h treatment

Conc. mg/ml viability (number of colonies) avg PE % Mutants (number of colonies) ∑M NPC MF/105cells Mt/Msc
NC (1) 335 402 394 377 102.8 2 2 2 6 4 5 4 4 6 1 36 2,764,667 1.30 1.09
NC (2) 337 364 368 356 97.2 1 1 6 1 6 5 3 2 1 2 28 2,613,111 1.07 0.90
0.01 (1) 298 311 328 312 85.2 2 4 3 3 1 4 3 3 2 3 28 2,290,444 1.22 1.03
0.01 (2) 304 312 387 334 91.2 3 2 2 4 3 4 3 1 5 4 31 2,451,778 1.26 1.06
0.015 (1) 379 378 376 378 103.0 3 4 2 6 2 2 4 1 4 4 32 2,769,556 1.16 0.97
0.015(2) 320 311 303 311 84.9 6 5 4 5 1 3 1 4 2 2 33 2,283,111 1.45 1.21
0.025 (1) 310 266 301 292 79.7 4 6 2 0 5 5 3 5 2 4 36 2,143,778 1.68 1.41
0.025 (2) 381 386 375 381 103.8 1 4 3 5 2 4 2 2 3 5 31 2,791,556 1.11 0.93
0.03 (1) 259 302 303 288 78.5 3 5 4 1 4 4 2 5 4 5 37 2,112,000 1.75 1.47
0.03 (2) 302 380 389 357 97.4 4 3 4 4 2 5 3 4 6 4 39 2,618,000 1.49 1.25
0.04 (1) 346 324 351 340 92.8 4 5 4 2 4 2 3 3 2 4 33 2,495,778 1.32 1.11
0.04 (2) 346 342 358 349 95.1 3 3 2 7 4 2 2 4 4 9 40 2,556,889 1.56 1.31
EMS50 320 328 286 311 84.9 40 46 45 40 51 41 38 46 39 47 433 2,283,111 18.97 15.94
EMS100 336 326 301 321 87.5 107 104 104 114 120 90 105 105 100 90 1 039 2,354,000 44.14 37.09

∑M = sum of mutants in all 5 dishes

NPC = no. of planted cells

MF/1E+05 cells = mutation frequency

Mt/Msc = no. of mutants in test conc. vs no. of mutants in solvent control

Mutagenicity with metabolic activation, 3h treatment

Conc. mg/ml Viability (number of colonies) avg PE % Mutants (number of colonies) ∑M NPC MF/1E+05 cells Mt/Msc
NC (1) 371 385 385 380 112.5 9 1 8 8 7 7 4 7 1 7 59 2,789,111 2.12 1.12
NC (2) 283 295 310 296 87.5 1 4 2 5 3 2 5 4 2 7 35 2,170,667 1.61 0.85
0.02 (1) 399 402 363 388 114.7 4 6 5 5 5 6 6 4 7 5 53 2,845,333 1.86 0.98
0.02 (2) 257 291 277 275 81.3 2 3 2 2 5 2 6 3 2 4 31 2,016,667 1.54 0.81
0.05 (1) 362 331 367 353 104.5 1 2 2 5 3 2 5 4 1 2 27 2,591,111 1.04 0.55
0.05 (2) 395 382 360 379 112.1 5 6 6 4 4 4 4 2 3 4 42 2,779,333 1.51 0.80
0.1 (1) 452 442 409 434 128.4 6 3 5 3 4 1 2 6 4 3 37 3,185,111 1.16 0.61
0.1 (2) 376 345 358 360 106.4 3 9 5 6 5 4 1 4 2 9 48 2,637,556 1.82 0.96
DMBA (1) 338 327 340 335 99.1 77 85 98 93 112 95 85 101 82 107 935 2,456,667 38.06 20.08
DMBA (2) 376 345 358 360 106.4 80 93 115 110 100 91 102 83 89 96 959 2,637,556 36.36 19.18

Mutagenicity with metabolic activation, 3h treatment

Conc. mg/ml Viability (number of colonies) avg PE % Mutants (number of colonies) ∑M NPC MF/1E+05 cells Mt/Msc
NC (1) 340 302 295 312 97.6 1 2 7 4 2 6 3 3 6 1 35 2,290,444 1.53 1.02
NC (2) 327 315 341 328 102.4 4 3 4 3 3 3 3 2 5 5 35 2,402,889 1.46 0.98
0.075 (1) 273 288 282 281 87.8 7 8 13 2 8 4 4 4 4 2 56 2,060,667 2.72 1.43
0.075 (2) 258 275 276 270 84.3 2 2 3 8 1 5 4 4 5 4 38 1,977,556 1.92 1.01
0.1 (1) 400 465 470 445 139.1 5 8 2 3 3 2 5 5 3 4 40 3,263,333 1.23 0.65
0.1 (2) 255 272 300 276 86.1 4 2 8 8 5 6 6 9 6 5 59 2,021,556 2.92 1.54
DMBA (1) 412 326 343 360 112.6 82 81 82 72 89 81 75 81 84 84 811 2,642,444 30.69 16.19
DMBA (2) 342 375 324 347 108.4 70 82 80 87 85 80 74 92 68 64 782 2,544,667 30.73 16.21

Applicant's summary and conclusion

Conclusions:
Test substance was not mutagenic for V79 cells with as well as without metabolic activation.
Executive summary:

Method

Test substance was assayed for mutagenicity in in vitro mammalian cell gene mutation test, according to OECD guideline 476.

Chinese hamster V79 lung fibroblasts were used for testing; test substance was dissolved in DMEM.

Cytotoxicity test without as well as with metabolic activation was performed in advance. Due to purchase optimal dose range, toxicity test was then repeated two times more. Concentrations used in overall cytotoxicity testing were in the range 0.0005 – 2.0 mg/ml.

On the basis of cytototoxicity testing results, six concentrations were chosen for the mutagenicity experiment without metabolic activation, i.e. 0.01, 0.015, 0.020, 0.025, 0.03 and 0.04 mg/ml. The concentration of 0.02 mg/ml was cancelled during the experiment for redundance.

Four concentrations 0.02, 0.05, 0.1 and 0.2 mg/ml were used for the experiment with metabolic activation. The concentration of 0.2 mg/ml was completely toxic and in the concentration 0.1 insufficient number of cells was transferred for further cultivation.

Additional experiment was performed with additional concentration 0.075 and repeated concentration 0.1 mg/ml to get four analysable concentration according to guidelines.

The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors.

Concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations.

Results

Average mutant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the experimental conditions, test substance was non-mutagenic for V79 cells with and without metabolic activation.