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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic.

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro Ames tests, in vitro gene mutation on mammalian cells and in vitro chromosomal aberration.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 3rd to November 11th, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The usage of information on Direct Blue 199_Na, which has the same main component and with a different counter ion, can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point.
The impurity profile does not impact on the read across proposed. Details on the approach followed are included in the document attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
ninth addendum, adopted July 21, 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Characteristics of the strains was checked every 2 to 6 months.
Histidine-auxotrophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet.
The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light. Strains containing the R-factor: TA 98 and TA 100, were additionally checked for ampicillin resistance.
Tryptophan-auxotrophy of E. coli WP2 uvrA was demonstrated by the requirement for tryptophan.
The absence of the uvrA gene was demonstrated by the sensitivity of the strain to UV-Iight.
All strains were checked for their characteristic reversion properties with known mutagens (reference item).

The strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10 ml + l ml) in a deep freezer at about -80 °C.
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9
Test concentrations with justification for top dose:
312.5, 625.0, 1250.0, 2500.0 and 5000.0 µg/plate, both preincubation assay and standard plate incorporation assay.
Vehicle / solvent:
- Vehicle/solvent bidistilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
other: 2-amonianthracene
Details on test system and experimental conditions:
TEST ITEM
On the day of the experiment, the test substance was dissolved in bidistilled water after warming up to 50°C in a water bath and strongly shaken. The test item was soluble up to the concentration of 50 mg/ml. Lower concentrations of the test item were obtained by serial dilution of the stock solution with bidistilled water. The stock solution was sterile filtered without residue.

PRE CULTURES
Aliquots from frozen stocks were grown in liquid nutrient broth medium (NB-medium, Oxoid, United Kingdom) for 8 hours and then used for the experiment. The bacterial cultures were incubated in a time-temperature controlled incubater at about 37 °C.

STANDARD PLATE INCORPORATION ASSAY - With and without metabolic activation
The plates with the selective agar (minimal agar plates) were made in-house. Each Petri dish contained about 20.0 ml of minimal agar (1.5 % agar supplemented with 2 % salts of the Vogel-Bonner Medium E and 2 % glucose). The agar used was Select AGAR, GIBCO BRL, Switzerland. Glucose, D (+) glucose, anhydrous. The Vogel-Bonner Medium E was prepared in-house.

Overlay Agar: the overlay agar contained per litre: 6.0 g of GIBCO BRL Select Agar and 6.0 g NaCl. Sterilisation was performed at 121 °C in an autoclave, cooled down to 50 °C and dispensed into glass bottles. On the day of test peformance, the agar was molten in a water bath and 10 % aliquots (v/v) of 0.5 mM L-histidine / 0.5 mM d-biotin for Salmonella strains or 0.5 mM tryptophan, dissolved in bidistilled water, for E. coli strains were added sterile filtered.

Plates: the following materials were mixed in a test tube and poured onto the minimal agar plates:
100 µI Test solution at each dose level, solvent (negative control) or reference item solution (positive control)
500 µ1 S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µI Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µI Overlay agar

PRE-INCUBATION ASSAY - With metabolic activation
In the pre-incubation assay 100 p1 test solution, 500 µl S9 mix and 100 µl bacterial suspension were mixed in a test tube and shaken at about 37 °C for 30 minutes. After pre-incubation 2.0 ml overlay agar (about 45 °C) was added to each tube and well mixed. The mixture was poured on minimal agar plates. In addition, the respective controls (solvent) and positive controls (reference item) were run together with each strain.
After solidification the plates were incubated upside down for at least 48 hours at 37° C ± 2 °C in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level including the controls, three plates were used.

MAMMALIAN MICROSOMAL FRACTION S9 Mix
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male rats (HanBrl:WIST SPF), delivered by RCC Ltd, Animal Breeding and Biotechnology, Füllinsdorf, Switzerland. The animals were treated with Aroclor 1254 (Analabs Inc., delivered from Antechnika, Karslruhe, Germany), 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCI. The homogenate was centrifuged for 15 minutes at 9000 × g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 42.5 mg/ml.

On the day of the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the mixture. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCI2
33 mM KCI
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium phosphate-buffer, pH 7.4.
Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator. The S9 mix preparation was performed according to Ames et al, 1983.

RANGE FINDING STUDY
To evaluate the toxicity of the test item a range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without metabolic activation at six concentrations of the test item. The concentrations applied were 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000.0 µg/plate. One plate was prepared per test item concentration, negative and positive controls. The plates were inverted and incubated for about 48 hours at 37 ± 2 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The range finding test is reported separately.

DATA RECORDING
Colonies were counted electronically using an Accucount 1000 (Biologics, Gainsville, Virginia, USA), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have interfered with automating counting. The results were sent on line to a computer. The operator checked them on a random basis. Observations indicating precipitates of the test item in the top agar or a reduced or absent bacterial background lawn were registered additionally.

ACCEPTABILITY of the ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if the mean colony counts of the negative contro) values of all strains are within the acceptable ranges listed in the historical data and if the results of the positive controls meet the criteria for a positive response. Normal bacterial background lawn shall be visible in the solvent control. In either case the final decision is based on the scientific judgement of the Study Director.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at oniy one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis was not required.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The plates incubated with the test item showed normal background growth up to 5000 pg/plate with and without S9 mix in both experiments. No reduction in the growth of the bacterial background lawn was observed. No precipitation of the test item was visible on the surface of the agar plates.
No substantial increase in revertant colony numbers of any of the five tester strains was observed at any concentration level, neither in the presence nor in the absence of an metabolic activation system (S9). There was also no tendency of higher mutation rates with increasing concentrations in the range beiow the generally acknowledged border of biological relevance.

Appropriate reference items were used as positive controls. They showed a distinct increase of induced revertant coionies.

Summary of the First Mutagenicity Test

Experiment with metabolic activation

Strain number TA 100 TA 1535 WP2 uvrA TA 98 TA 1537
µg/plate Mean Mean Mean Mean Mean
Bidist. water 122 22 35 31 17
312.5 106 23 38 29 11
625.0 107 22 34 30 8
1250.0 102 19 34 36 13
2500.0 100 22 31 30 15
5000.0 94 22 33 31 10
2-A-anthracene 1.5 1548 1084 264
2-A-anthracene 20.0 858
CPA 200.0 324

Experiment without metabolic activation

Strain number TA 100 TA 1535 WP2 uvrA TA 98 TA 1537
µg/plate Mean Mean Mean Mean Mean
Bidist. water 122 23 30 23 10
312.5 119 28 26 28 13
625.0 114 25 29 25 11
1250.0 129 26 30 22 8
2500.0 124 31 26 19 9
50000.0 114 25 24 23 8
2-Nitrofluorene 5.0 583
4-Nitroquinoline 2.0 865
9-A-acridine 80.0 1191
Sodium azide 2.0 862 720

Summary of the Second Mutagenicity Test

Experiment with metabolic activation

Strain number TA 100 TA 1535 WP2 uvrA TA 98 TA 1537
µg/plate Mean Mean Mean Mean Mean
Bidist. water 101 16 36 40 14
312.5 107 20 29 34 8
625.0 104 19 40 25 15
1250.0 106 22 39 28 13
2500.0 101 16 32 20 12
5000.0 107 21 36 17 14
2-A-anthracene 1.5 1171 925 255
2-A-anthracene 20.0 471
CPA 200.0 367

Experiment without metabolic activation

Strain number TA 100 TA 1535 WP2 uvrA TA 98 TA 1537
µg/plate Mean Mean Mean Mean Mean
Bidist. water 108 24 20 18 8
312.5 106 20 23 15 6
625.0 109 21 28 16 7
1250.0 102 21 25 22 9
2500.0 100 23 26 19 10
5000.0 105 17 20 20 5
2-Nitrofluorene 5.0 725
4-Nitroquinoline 2.0 779
9-A-acridine 80.0 1014
Sodium azide 2.0 874 683
Conclusions:
The test substance did not induce gene mutation by base-pair changes or frameshifts, in the Salmonella typhimurium and Escherichia coli reverse mutation assay, under the experimental conditions reported.
Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test ( first experiment with and without metabolic activation, second experiment without activation) using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, the negative (solvent) and the positive controls (reference items) were tested in triplicate. The test item was tested at the following concentrations: 312.5, 625.0, 1250.0, 2500.0 and 5000.0 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No reduction in the growth of the bacterial background lawn was observed. No precipitation of the test item was visible on the surface of the agar plates. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any concentration level, neither in the presence nor in the absence of an metabolic activation system (S9). There was asso no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference items were used as positive controls. They showed a distinct increase of induced revertant coionies.

Conclusion

Under the test conditions, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 18th to June 06th, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: B.49, In Vitro Mammalian Cell Micronucleus Test, Commission Regulation (EU) No. 640/2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
The human peripheral blood lymphocytes used for testing were obtained from healthy non smoking females (up to 35 years of age).
Peripheral blood (heparinized) is taken from donors in certified medical laboratory (MeDiLa) in the morning and transported into the test facility as soon as possible
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
First experiment: 500, 1000 and 2000 μg/ml
Second experiment: 250, 500, 1000 µg/ml
Untreated negative controls:
yes
Positive controls:
yes
Remarks:
aneugenicity control (colchicine) serves as the positive control without S9-mix, and the clastogenicity control (cyclophosphamide) is used to test of the adequacy of the metabolic activation system used
Positive control substance:
cyclophosphamide
other: colchicine
Details on test system and experimental conditions:
TREATMENT AND CULTIVATION
50 µl of an appropriate concentration of test substance solution in medium was added to lymphocyte culture (2.5 ml growth medium RPMI-M + cca 150-210 µl human peripheral blood) in the presence and absence of a metabolic activation system (S9-mix).
In the first experiment after shaking the cultures were cultivated with the test substance for 3 hours. After that they were rinsed up by RPMI-M and then transported to the fresh growth medium (RPMI-M) with 11.25 µl of cytochalasin B (the final concentration in cultures was 4.5 µg/ml). Cultures were then cultivated and sampled after 23 hours since the beginning of treatment. The first experiments gave negative results, so the second experiment without metabolic activation had to be done with extended exposure.
In the second experiment (prolonged exposition without activation) cultures were treated with test substance without metabolic activation for 23 hours in the presence of cytochalasin B (7.5 µl; final concentration in cultures was 3 µg/ml). No transport to fresh medium was needed in that experimental design.

REPLICATES: duplicate cultures were used for each concentration and control.

CELL CYCLE LENGTH DETERMINATION
The proliferation of lymphocytes was evaluated by doubling time experiment. Four separate tubes with blood in the medium were prepared in day 2. Every following day (day 3, day 4 and day 5) lymphocytes were isolated and quantified. Then the proliferation curve was constructed and cell cycle length was determined. The lymphocyte`s cell cycle length during the first experiment was 12 hours.

SCHEDULE OF THE TEST
Day 1: blood sampling. Blood was taken and then stored in the fridge until the next day.
Day 2 and 3: cultivation. The whole human peripheral blood was transferred to the growth medium and mitogenic stimulator (phytohaemagglutinin M) was added. These operations were carried out in a laminar box at room temperature. The cultivation runs without interrupting for 48 hours (37 ± 1 °C; 5 % CO2).
Day 4: exposition. The test substance, positive and negative control substances were added to the individual cultures (in a laminar box at room temperature). In the first experiment without and with metabolic activation (S9-mix), the cultures were washed (after 3 hours of continuous exposure to the test substance in 37 ± 1 °C; 5 % CO2) and transferred to fresh culture medium with cytokinesis blocker (cytochalasin B). Washing and transfer were carried out in a laminar box at room temperature. Then the lymphocytes were cultured (37 ± 1 °C; 5 % CO2) for remaining period (total 23 hours from the start of exposure).
Day 5: harvesting of cultures. All cultures were processed in a laminar box at room temperature, (with hypotonia, fixation solution). Suspensions were dropped on clear microspcopic slides. The slides were allowed to dry at least overnight.
Day 6: staining of slides. Slides were stained by Giemsa-Romanowski staining solution.

PREPARATION OF SLIDES
Cultures were harvested 23 hours after the beginning of treatment (after about 1.5 to 2 cell cycles). Cultures were treated by hypotonic solution (RT, ca 5 min.) and then they were centrifuged (1200 rpm, 10 min.). After removing of hypotonic solution, fixation solution was added to cultures and cultures were centrifuged again (1200 rpm, 10 min.). The addition of fixation solution and centrifugation were repeated three times. Suspensions were then dropped on clear microscopic slides. Preparations were let to dry at laboratory temperature and then slides were stained by Giemsa Romanowski staining solution.

SCORING OF CELLS FOR CYTOTOXICITY AND GENOTOXICITY EVALUATION
All slides were coded before microscopic analysis.
At least 1000 cells were scored per each concentration and controls divided equally between the duplicates for determination of cytotoxicity. CBPI index was calculated from ratio of mononucleated, binucleated and multinucleated cell at each culture. The cytotoxic effect was characterized as % of cytotoxicity.
2000 binucleated cells were analysed per each concentration and control divided equally between the duplicates for determination of genotoxicity. The genotoxic effect is characterized by numbers of binucleated cells with micronuclei.

ACCEPTABILITY OF EXPERIMENT RESULTS
Results are accepted if:
- numbers of binucleated cells with micronuclei in negative and positive controls are in actual ranges of historical controls
- at least 1000 binucleated cells per culture are available for scoring at each concentration (if there is clearly positive results, the number of scoring cells could be lower).
- at least 3 concentrations are analysable.

DOSE SELECTION
At first the cytotoxicity of test substance has been determined by measuring of cell proliferation. In the highest concentration (2000 µg/ml) no cytotoxicity or precipitate was observed; test item did not cause a marked change in the pH of the medium (in used concentrations).
Cytotoxicity test concentrations:
First experiment 125, 250, 500, 1000 and 2000 µg/ml
Second experiment 250, 500, 1000 and 2000 µg/ml
The highest concentration 2000 µg/ml could not be used for genotoxicity evaluation because of high cytotoxicity in this exposure time (23 hours).

METABOLIC ACTIVATION SYSTEM
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 (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 (1983). 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. Fresh solution of the cofactors (1.6 mM MgCl2.6H2O, 0.8 mM NADP and 1 mM glucose-6-phosphate) was prepared before each experiment with metabolic activation. Each culture in experiments with metabolic activation contained 18.5 µl of S9 and 18.5 µl of cofactors solution (S9-mix).
Evaluation criteria:
Genotoxic potential is indicated by increasing of number of binucleated cells with micronuclei in comparison to the negative control (two-fold increase rule) and by dependence of number of binucleated cells with micronuclei on dose (dose-response relationship).
Ratio of number of binucleated cells with micronuclei at tested concentration to number of binucleated cells with micronuclei in negative control (Mt/Mc) should be higher than 2. The result will be considered as positive when two-fold increase rule will be met at least one tested concentrations.
Test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control (two-fold increase rule)
- the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship) is evident
- any of the results are outside the distribution of the historical negative control data

Test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data
In case of equivocal results, further testing with modification of experiment conditions will be used for clarification.
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The first experiments gave negative results, so the second experiment without metabolic activation had to be done with extended exposure (23 hours) in the presence of cytochalasin B. The results did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (Mt/Mc > 2, two-fold increase).

Actual results of negative and positive controls fulfilled the acceptability criteria - the actual values did not exceed limits of historical controls in the testing laboratory.

CYTOXICITY
All of test concentrations did not show the cytotoxicity higher than 55 ± 5 % in the time of exposure 3 hours. Test concentration 2000 µg/ml did show the cytotoxicity in the prolonged exposition without activation (23 hours). Other used concentrations 1000, 500 and 250 µg/ml did not show the cytotoxicity in the prolonged exposition.
On the basis of these results, the concentration of 2000 µg/ml was selected as the highest concentration for the analysis of genotoxic effect in the time of exposure 3 hours. For the analysis of genotoxic effect in the prolonged time of exposure 23 hours was selected as the highest concentration 1000 µg/ml (second experiment).
Conclusions:
Under the experimental design described, the test substance had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.
Executive summary:

In Vitro Mammalian Cell Micronucleus Test was performed according to OECD Test Guideline No. 487.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in RPMI medium and assayed in five concentrations 125 - 2000 µg/ml, which were applied to cultures in volume of 50 µl.

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

Under the experimental design described, the test substance had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Conclusion

Under the experimental design described, the test substance had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 13th to April 07th, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 28th, 2015
Deviations:
yes
Remarks:
no impacting the outcome of the study
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
no impacting the outcome of the study
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Source: frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC).
- Breeding: the cells were kept at -196 ºC under liquid nitrogen. After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS in incubator (5 % CO2, 37±1 °C, moistened). Cells underwent maximum 7 passages after thawing the original culture delivered from cell collection before using for test.
- Cleaning: cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants.
- Mycoplasma determination: cell cultures were checked for mycoplasma contamination. At every experiment one withdrawal of media has been performed and sent to the contract laboratory performing mycoplasma determination.
- Cell cycle duration: duration of cell cycle was determined according to ATCC Animal Cell Culture Guide, Tips and techniques for continous cell lines.)
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:
EXPERIMENT: 0.25, 0.5, 1.0, and 2.0 mg/ml
CYTOTOXICITY: 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/ml
Vehicle / solvent:
The test substance is soluble in water so it was tested dissolved in Dulbecco's minimal essencial medium (DMEM).
Untreated negative controls:
yes
Remarks:
negative control plates contained 10 ml of complete medium
Negative solvent / vehicle controls:
yes
Remarks:
plates (DMSO) contained 9.9 ml of completemedium and 0.1 ml of DMSO
Positive controls:
yes
Remarks:
plates contained 9.9 or 9.95 ml of complete medium and 100 or 50 µl of relevant positive control diluted in DMSO
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
APPLICATION
Every dish with negative control/test substance contained 1.0 ml of application form of the test substance in DMEM so that the final concentrations on dishes were as given above and 9.0 ml of complete medium or activation mixture (S9 mix).

DURATION and SCHEDULE
- Treatment: cells were treated for 3 hours (with as well as without metabolic activation; day 1).
- Seeding: after treatment, approximately 2x10^6 cells were transferred to suitable number of dishes to seed enough cells.
- Determination of plating efficiency: cells were seeded for detection of number of cells (PE estimation).
- Subculturing: on the 3rd, 6th and 8th day, approximately 2x10^6 cells from every culture were transferred and 10th day. The cell populations were subcultured in order to maintain them in exponential growth.
- Extraction of mutants: extractions of mutants was performed with using selective medium together with PE estimation again.

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 two millions of viable cells. Cells were grown in 10 cm Petri dishes.

INCUBATION
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.

NUMBER OF REPLICATIONS
Every concentration was tested in two independent runs.

DETERMINATION OF MUTANT FREQUENCY
At expression time, 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 10th day.
- after dilution, an estimated 220000 cells were plated in each of ten 100 mm tissue culture Petri dishes (together 2200000 cells). After 1 hour, 6-thioguanine was added to each the 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.

ASSSAY ACCEPTANCE CRITERIA
- Concurrent negative controls should be within the 95 % control limits of the distribution of the laboratory’s historical negative control database.
- Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
- Two experimental conditions (i.e., with and without metabolic activation) were tested unless one resulted in positive results.
- Adequate number of cells is used and concentrations are analysable.


CYTOTOXICITY
The test substance was dissolved in DMEM at the maximum concentration of 20.0 mg/ml. This concentration was dissolved according to recommendation given in OECD TG 476 to three other lower concentrations. These concentrations were 2.5, 5.0, and 10.0 mg/ml.

METABOLIC ACTIVATION SYSTEM
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. Composition of S9 mix was as follows:
S9 tissue fraction 1.0 ml
NADP (0.1M) 0.4 ml
G-6-P (0.1M) 0.5 ml
KC1 (0.33M) 1.0 ml
MgCl2 (0.1M) 0.5 ml
Phosphate Buffer (0.2 M) 4.6 ml
Each plate in all experiments with metabolic activation contained 4 ml of S9mix, 5 ml of complete medium and 1.0 ml of the test substance solution.
Evaluation criteria:
Each experiment is separately evaluated 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 the 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 repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions i.e. S9 concentration or S9 origin) could be performed.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MUTAGENICITY
Mutation frequency of cells treated with the test substance in all experiments was low in all concentrations; Mt/Msc ratio generally did not exceeded 3 fold limits. At the same time, no dose dependence was observed in any experiment.
As nor the dose-dependence neither repeated 3-fold increase was reached we do not consider these single increased values biologically significant.

Mutation frequency of negative controls varied from 0.77 – 1.53 mutants per 10^5 plated cells. Historical control range is 0.0-3.42 mutants per 10^5cells.
Mutation frequency of positive controls was sufficiently high:
EMS 50 µl 11.14, historical control range is 6.49 – 21.75 mutants per 10^5 cells
EMS 100 µl 15.10, historical control range is 12.43 – 46.34 mutants per 10^5 cells.
DMBA 19.91 and 22.68, historical control range is 0 – 46.34 mutants per 10^5 cells

PLATING EFFICIENCY
For the assessment of number of plated cells, PE was determined always after treatment. In all concentrations used in both experiments more than two millions cells were influenced in single replicates.
In most cases more than two millions of cells were grown for expression of mutants. It does not apply to treatment with metabolic activation 2.0 mg/l, replicate 2, where less than two million cells were transferred to further growth after treatment. Another such case was the dose of 1.0 mg/ml in experiment with metabolic activation (replicate 1) where 3 dishes in the last passage were contaminated with mould. In this case only cells from the other 4 dishes were processed. Both the doses are marked with cursive letters.

CYTOTOXICITY
Treatment with the test substance for 3 hours resulted in no toxicity.
On the basis of the result obtained, the concentration of 0.5 mg/ml was selected as the highest concentration to be used in the mutation assays-. The lower concentrations were spaced by a factor approximately 2-√10, in order to reach a concentration row.

MYCOPLASMA DETERMINATION
Results of both test samples were negative so all media after cultivation of cells were free of mycoplasma.

MUTAGENICITY

Summary of results: 3-hour treatment without metabolic activation

Conc. (mg.ml) MF/105 Mt/Msc
NC 0.88 1.00
0.25 0.74 0.84
0.5 0.5 0.57
1 0.68 0.77
2 0.75 0.85
EMS50 11.14 12.6
EMS100 15.1 17.08

Summary of results: 3-hour treatment with metabolic activation

Conc. (mg.ml) MF/105 Mt/Msc
NC 1.33 1.00
0.25 1.38 1.04
0.5 1.24 0.93
1 1.17 0.88
5 0.73 0.55
DMBA 21.14 15.93

Mutagenicity without metabolic activation, 3-hour treatment - extraction time 9 days

Conc. (mg/ml) Viability (number of colonies) avg PE (%) Mutants (number of colonies) ∑M NPC MF/105 cells Mt/Msc
NC1 439 437 398 425 102 2 2 6 2 4 3 2 4 2 4 31 3114222 1 1.13
NC2 440 366 419 408 98 4 4 1 2 1 1 3 2 3 2 23 2994444 0.77 0.87
0.25 (1) 445 432 459 445 106.9 0 3 3 3 4 6 2 2 2 0 25 3265778 0.77 0.87
0.25 (2) 485 493 492 490 117.6 5 3 2 2 0 3 2 3 2 4 26 3593333 0.72 0.82
0.5 (1) 358 415 392 388 93.2 2 2 2 0 1 2 4 0 0 0 13 2847778 0.46 0.52
0.5 (2) 424 413 373 403 96.8 1 1 3 2 1 1 2 1 3 1 16 2957778 0.54 0.61
1.0(1) 438 445 483 455 109.3 1 1 4 0 2 2 1 1 2 4 18 3339111 0.54 0.61
1.0(2) 345 368 396 370 88.8 4 1 3 3 2 4 1 2 2 1 23 2710889 0.85 0.96
2.0(1) 375 375 326 359 86.1 3 3 1 2 1 1 3 1 0 3 18 2630222 0.68 0.77
2.0(2) 336 307 306 316 76.0 2 1 3 1 3 1 1 3 3 1 19 2319778 0.82 0.93
EMS50 343 325 346 338 81.2 18 27 33 31 22 26 36 32 22 29 276 2478667 11.14 12.6
EMS100 507 525 507 513 123.2 51 47 53 67 57 61 62 63 59 48 568 3762000 15.1 17.08

Mutagenicity with metabolic activation, 3-hour treatment - extraction time 9 days

Conc. (mg/ml) Viability (number of colonies) avg PE (%) Mutants (number of colonies) ∑M NPC MF/105cells Mt/Msc
NC1 335 336 362 336 92.2 3 3 3 1 4 2 1 5 1 4 27 2460333 1.1 0.83
NC2 363 365 423 384 105.4 4 5 1 5 3 3 5 6 7 4 43 2813556 1.53 1.15
0.25 (1) 363 372 375 370 101.6 1 4 4 3 1 5 4 3 4 0 29 2713333 1.07 0.81
0.25 (2) 276 294 300 290 79.7 4 3 3 5 3 2 1 6 6 5 38 2126667 1.79 1.35
0.5 (1) 339 308 325 324 89 3 4 2 1 1 1 6 2 4 1 25 2376000 1.05 0.79
0.5 (2) 271 288 289 283 77.7 4 6 3 3 1 2 1 2 3 5 30 2072889 1.45 1.09
1.0(1) 374 367 365 369 101.3 3 4 2 3 4 3 2 4 1 2 28 2703556 1.04 0.78
1.0(2) 370 421 384 392 107.6 7 3 0 3 3 4 2 4 5 6 37 2872222 1.29 0.97
2.0(1) 386 407 384 392 107.8 2 3 4 2 4 3 6 1 2 2 29 2877111 1.01 0.76
2.0(2) 384 435 429 416 114.3 1 2 0 1 0 4 2 1 2 1 14 3050667 0.46 0.35
DMBA 373 408 378 386 106.1 60 52 42 39 51 69 70 74 50 57 564 2833111 19.91 15
DMBA 326 311 290 309 84.9 51 55 57 65 49 60 59 41 38 39 514 2266000 22.68 17.09

PLATING EFFICIENCY

Plating efficiency after 3-hour treatment and number of survived cells – experiment without metabolic activation

Conc. (mg/ml) Viability (number of colonies) mean PE (%) ∑ NSC NPC ∑ NPC
NC1 307 269 283 286 96.9 8.47E+06 2.34E+06 4.83E+06
NC2 274 303 337 305 103.1 7.27E+06 2.49E+06
0.25 (1) 278 242 279 266 90.1 1.01E+07 2.18E+06 4.27E+06
0.25 (2) 270 239 260 256 86.7 1.11E+07 2.09E+06
0.5 (1) 287 250 254 264 89.2 8.53E+06 2.15E+06 5.18E+06
0.5 (2) 396 360 356 371 125.4 7.13E+06 3.03E+06
1.0(1) 300 312 282 298 100.8 1.01E+07 2.43E+06 4.99E+06
1.0(2) 295 296 347 313 105.8 8.00E+06 2.55E+06
2.0(1) 374 402 363 380 128.5 1.22E+07 3.10E+06 6.27E+06
2.0(2) 357 411 396 388 131.3 8.80E+06 3.17E+06
EMS50 858 850 910 873 295.3 1.17E+07 7.13E+06 -
EMS100 304 326 367 332 112.5 9.27E+06 2.71E+06 -

Plating efficiency after 3-hour treatment and number of survived cells – experiment with metabolic activation

Conc. (mg/ml) Viability (number of colonies) mean PE (%) ∑ NSC NPC ∑ NPC
NC1 261 249 255 255 93.5 4800000 2080000 4450000
NC2 297 284 290 290 106.5 3870000 2370000
0.25 (1) 258 220 278 252 92.4 7930000 2060000 4550000
0.25 (2) 279 321 315 305 111.9 7000000 2490000
0.5 (1) 250 222 250 241 88.3 7400000 1970000 4020000
0.5 (2) 250 253 250 251 92.1 6900000 2050000
1.0(1) 261 270 276 269 98.7 7150000 2200000 4420000
1.0(2) 246 291 281 273 100 7300000 2230000
2.0(1) 248 244 274 255 93.6 8600000 2090000 3760000
2.0(2) 218 217 181 205 75.3 7600000 1680000
DMBA 255 264 281 267 97.8 5800000 2180000 4180000

CYTOTOXICITY

Toxicity test without metabolic activation, 3-hour treatment

Concentration (mg/ml) Viability (number of cells per plate) avg PE
DMEM 332 337 347 339 100
0.0625 466 475 500 480 141.8
0.125 422 456 415 431 127.3
0.25 331 304 321 319 94.1
0.5 366 351 330 338 99.8
1 332 356 297 328 96.9
2 365 342 397 368 108.7
Conclusions:
Under the experimental conditions, the test substance was non-mutagenic for V79 cells with as well as without metabolic activation.
Executive summary:

The test substance was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The performed test was based on OECD Test Guideline No. 476 – In Vitro Mammalian Cell Gene Mutation Test (2015), which is analogous to the EU method B.17. V79 hamster fibroblast were used for testing. The test substance was dissolved in DMEM in concentration 2.0 mg/ml. Concentrations tested for cytotoxicity in 3-hour experiment were 2.0, 1.0., 0.5, 0.25, 0.125 and 0.0625 mg/ml. No toxicity was observed in any dose.

Mutagenicity experiment (3 hour treatment) with the test substance followed then. Four concentrations of test substance - 2.0, 1.0, 0.5 and 0.25 mg/ml - were used. Each concentration was tested in two independent runs. Experiments were performed with as well as without metabolic activation.

In the arrangement given above, the test substance was non-mutagenic for V79 cells without as well as with metabolic activation.

Conclusion

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro Ames tests, in vitro gene mutation on mammalian cells and in vitro chromosomal aberration.

 

BACTERIA GENE MUTATION ASSAY

In vitro bacteria gene mutation assay were evaluated taken into account the already existing data on Direct Blue 199, sodium salt (i.e. Direct Blue 199_Na). The usage of information on Direct Blue 199_Na, which has the same main component and with a different counter ion, can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point. The impurity profile does not impact on the read across proposed. Details on the approach followed are included in the document attached to the IUCLID section 13.

 

Direct Blue 199_Na was assessed for its potential to induce gene mutations according to the plate incorporation test (first experiment with and without metabolic activation, second experiment without activation) using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No reduction in the growth of the bacterial background lawn was observed. No precipitation of the test item was visible on the surface of the agar plates. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any concentration level, neither in the presence nor in the absence of an metabolic activation system (S9). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

A second experiment was conducted on five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA1538) of Salmonella typhimurium both in the absence and presence of metabolic activation. No toxicity or precipitation of test agent was observed in the first experiment, and at the sponsor's request, the same doses were used for all experiment 2 treatments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges and were significantly elevated by positive control treatments. No test substance treatment of any of the test strains induced a significant increase in revertant numbers. In no case was there a two-fold (strains TA98 and TA100) or three-fold (strains TA1535, TA1537 and TA1538) increase in revertant numbers that would normally be required for a clear induction of mutation in these strains.

 

MAMMALIAN CELL GENE MUTATION ASSAY

Direct Blue 199 was assayed for the mutagenicity following the testing procedures outlined in the OECD Guideline No. 476. V79 hamster fibroblast were used for testing and the concentrations tested for cytotoxicity in 3-hour experiment were 2.0, 1.0., 0.5, 0.25, 0.125 and 0.0625 mg/ml. No toxicity was observed in any dose. Mutagenicity experiment (3 hour treatment) with the test substance followed then; four concentrations of test substance - 2.0, 1.0, 0.5 and 0.25 mg/ml - were used. Each concentration was tested in two independent runs. Experiments were performed with as well as without metabolic activation. The test substance was non-mutagenic for V79 cells without as well as with metabolic activation.

 

CHROMOSOMAL ABERRATION ASSAY

In Vitro Mammalian Cell Micronucleus Test was performed according to OECD Test Guideline No. 487 on Direct Blue 199. The human peripheral blood lymphocytes from healthy donors were used for testing. Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors. Under the experimental conditions, the test substance had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation. The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).