Reaction mass of 2,2'-(vinylenedi-p-phenylene)bis[5-methylbenzoxazole] and 2,2'-(vinylenedi-p- phenylene)bisbenzoxazole and 2-[4-[2-[4-(benzoxazol-2- yl)phenyl]vinyl]phenyl]-5-methylbenzoxazole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 1535, TA 1537, TA 1538, TA 98

Details on mammalian cell type (if applicable):

Bacteria were grown overnight in nutrient broth (25 g oxid Nurrient Broth No 2/ litre) at 37 °C. The suitable amount of bacteria in the cell suspension was checked by nephelometry. For incubation, stock cultures which were stored at - 80 °C were used.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Bacteria were grown overnight in nutrient broth (25 g oxid Nurrient Broth No 2/ litre) at 37 °C. The suitable amount of bacteria in the cell suspension was checked by nephelometry. For incubation, stock cultures which were stored at - 80 °C were used.

Metabolic activation:

with and without

Metabolic activation system:

rat Iiver homogenate fraction (S9)

Test concentrations with justification for top dose:

Main experiment: 4, 20, 100, 500, 2500 and 5000 µg/plate
Toxicity experiment and dose range finding: 4, 20, 100, 500, 2500 and 10000 µg/plate

Vehicle / solvent:

- Solvent: DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: N-Methyl-N'-nitro-N-nitrosoguanidine // 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
Top Agar was prepared for the salmonella strains by mixing 100 ml agar (0.6 % NaCl) with 5 ml of a 1.0 mM histidine and 5 ml of 1.0 mM biotin solution.
In the case of E. coli histidine was replaced by tryptophan (5 ml, 0.5 mM).
The following ingredients were added (in order) to 2 ml of molten top agar at 45 °C: 0.1 ml test compound solution; 0.1 ml of an overnight broth culture of the bacterial tester strain; 0.5 ml S9 Mix (if required) or buffer.
After mixing, the liquid was poured into a petri dish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose).

INCUBATION
Incubation for 48 to 72 hours at 30 °C in the dark.

NUMBER OF REPLICATIONS
3 plates per dose.

METABOLIC ACTIVATION
The requirement for metabolic activation was investigated by incorporating into the test an activation system by nicotinamide-adenine dinucleotia phosphate (NADP+)-cytochrome P450 dependent mixed function oxidase enzymes of the liver. The 9000 g supernatant of rat liver homogenate has been shown to be very useful in metabolic activation of foreign compounds. The animals were pretreaied with Aroclor 1254 as an inducer of several drug metabolizing enzymes.

Preparation and storage of Iiver homogenate fraction (S9)
Male Sprague Dawley rats (200 - 300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw) 5 days before sacrifice. Preparation was performed at 0 to 4 °C using cold sterile solutions and glassware. The liver from at least 5 - 6 animals were removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers). The washed livers were cut into small pieces and homogenised in three volumes of KCl. The homogenate was centrifuged at 9000 g for 10 minutes. The supernatant was the S-9 fraction. It was divided into small portions, rapidly frozen and stored at -80 °C for not longer than 3 months.

Preparation of S9 Mix and concentration of cofactors
Sufficient S9 fraction was thawed immediately before each test at room temperature. One volume of the S9 fraction were miixed with 9 volumes of the S9 co-factor solution and kept on ice until used. The concentrations of the different compounds in the S9 Mix were:
8 mM MgCl
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP+
100 mM phosphate buffer pH 7 ,4

TOXICITY EXPERIMENT
- Preparation: 0.1 ml of the different dilutions of the test compound were thoroughtly mixed with 0.1 ml 10^-6 dilutions of the overnight culture of TA 100 and plated with histidine and biotin rich top agar.
- Replicates: 3 plates per dose.

DOSE RANGE FINDING
- Strains: preliminary toxicity tests were performed with all tester strains.
- Indication of toxicity: a reduced rate of spontaneously occurring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopicaly.

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA 1537, TA 1538, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test compound did not cause a significant increase in the number of colonies with any of the tester strains in the absense or presence of S9 mix. No dose dependent effect was obtained.

TOXICITY TEST
The test compound was tested at doses of 4 to 10000 µg/plate and proved to be not toxic. Visible precipitation on the plates has been observed at 500 µg/plate. Thinning of the bacterial lawn or a reduction in the number of colonies have not been observed.
For mutagenicity test, 5000 µg/plate was chosen as the highest dose.

STERILITY CHECKS AND CONTROL PLATES
Sterility of S9 mix and the test compound was indicated by the absence of contamination on test material and S9 mix sterility check' plates. Control plates (background control and positive controls) gave the expected number of colonies.

Conclusions:

The substance resulted to be not mutagenic in bacterial test systems neither with nor without exogenous metabolic activation.

Executive summary:

The substance was tested for mutagenicity with strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 µg/plate to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

The test compound proved to be not toxic to the bacteria. 5000 µg/plate was chosen -as top dose level for the mutagenicity study.

In the absence of the metabolic activation system the test compound did not show a dose dependent influence on the number of revertants in any of the bacterial strains. Also in the presence of metabolic activation system, treatnent of the cells with test item did not result in relevant increases in the number of revertant colonies.

Conclusion

The substance resulted to be not mutagenic in bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 18 to February 05, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 29th July, 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cells Cultures).
- Suitability of cells: these cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h).
- Storage: the cell stocks were kept in liquid nitrogen.
- Seeding: cells were propagated from stock cultures, seeded in culture medium at a density such that the cells in suspensions continued to grow exponentially until harvest time.

MEDIA USED
- Incubation: the laboratory cultures are maintained in 75 cm2 plastic flasks at 37 °C in an incubator with a humidified atmosphere, set at 5 % CO2.
- Properly maintained: the V79 cells for the study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM and 1 % of Antibiotic-antimycotic solution (containing 10000 IU/ml penicillin, 10 mg/ml streptomycin and 25 μg/ml amphoptericin-B)) and heat-inactivated bovine serum (final concentration 10 %).
- Periodically checked for Mycoplasma contamination: checking for mycoplasma infections was carried out before freezing. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA × 4 Na) solution is used for cell detachment to subculture.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time 250, 500, 1000 and 2000 μg/ml test item without S9 mix and 125, 250, 500 and 1000 μg/ml test item with S9 mix
Experiment B with 20/20 h treatment/sampling time 62.5, 125, 250 and 500 μg/ml test item without S9 mix
Experiment B with 20/28 h treatment/sampling time 62.5, 125, 250 and 500 μg/ml test item without S9 mix
Experiment B with 3/28 h treatment/sampling time 125, 250, 500 and 1000 μg/ml test item with S9 mix

Vehicle / solvent:

- Colventicle: DMSO
- Justification for choice of solvent/vehicle: the solvent was compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test. The suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

PROCEDURES
EXPERIMENT A - 3-hour treatment, harvest 20 hours from the beginning of treatment.
- Exposure: after 24 hours, the culture medium of exponentially growing cell cultures was replaced with DME medium containing 5 % bovine and the test item.
- Exposure period: 3 hours at 37 °C.
- Post exposure: washing the cells with medium followed the exposure period and then growth medium was added.
- Sampling: sampling wase made at 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment).

EXPERIMENT B - 20-hour treatment, harvest 20 hours from the beginning of treatment
- Exposure: during the exposure period the cells were covered with medium containing the test item.
- Exposure period: the exposure period without metabolic activation was 20 hours; the exposure period with metabolic activation was 3 hours.
- Post exposure: the exposure period was followed by washing the cells with DME medium and growth medium was added.
- Sampling: sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

REPLICATES: duplicate cultures were used at each concentration and the solvent control culture as well as the positive controls for treatment without and with S9 mix. 5 × 10^5 cells/dish were set up for each group.

MEASUREMENT OF pH AND OSMOLARITY
The pH and osmolality of the each one treatment solution was measurement start the treatment.

PREPARATION OF CHROMOSOMES
Cell cultures were treated with Colchicine 2-2.5 hours prior the harvesting. These cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (3:1 mixture of methanol:acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried.

DETERMINATION OF CYTOTOXICITY
In order to determine the treatment concentrations of test item in the cytogenetic study a pre-experiment on cytotoxicity was performed. During the cytotoxicity assay 1-3 day old cultures was prepared in DME medium.
Cells were seeded into 92 × 17 mm dishes at 5 × 10^5 cells each and were incubated for 24 hours in 10 ml of DME medium containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 μl/ml) and were incubated at 37 °C for 3 hours. After treatment the cultures were washed with DME medium and covered with DME containing 10 % foetal bovine serum. Cell counts were performed after 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment). The cells were counted using a Bürker chamber. Additional groups of cells were treated for 20 hours without metabolic and for 3 hours with metabolic activation, with cell counts conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix).
Additionally, 4 cultures were set up for determining the initial cell count.
Based on the cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity.
The volume of culture medium was 5 ml/dish for each group.

PRELIMINARY SOLUBILITY TEST
A non GLP preliminary solubility test was performed suspending the test item in DMSO. A homogeneous suspension was obtained up to a concentration of 100 mg/ml.

METABOLIC ACTIVATION FRACTION
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2, D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier. The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.
The concentration of S9 was 1.5 % in medium.
The complete S9 Mix was freshly prepared before starting the experiments, containing components with the following ratios: S9 fraction 3 ml, HEPES 20 mM 2 ml, KCl 330 mM 1 ml, MgCl2 50 mM 1 ml, NADP 40 mM 1 ml, Glucose-6-phosphate 50 mM 1 ml, DME medium 1 ml.
Before adding to the culture medium the S9 Mix was kept in an ice bath.

ACCEPTANCE CRITERIA
The Chromosome Aberration Assay is considered acceptable if it meets the following criteria:
- the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data,
- concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control,
- cell proliferation in the solvent control is adequate,
- adequate number of cells and concentrations are analyzable,
- all requested experimental conditions are tested unless one resulted in a positive result,
- the criteria for the selection of the top concentration are fulfilled.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item 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,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item 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 laboratory historical negative control data.
Both biological and statistical significance should be considered together.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control.
The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.

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

Positive controls validity:

valid

Additional information on results:

In both experiments, clear cytotoxicity of about 50 % was observed at the highest concentration of 2000 µg/ml after test item 3 hours treatment in the absence and presence of metabolic activation. After 20-hours treatment, ca 50 % of cytotoxicity was recorded at 250 µg/ml in plates without S9 mix.
No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.
In the experiment B in the absence of metabolic activation one value at the dose of 62.5 µg/ml and in the presence of metabolic activation one value at the dose of 1000 µg/ml (5 aberrant cells excluding gaps/150 cells) were slightly above the 95 % control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells).
However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant.
No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of test item.

SOLUBILITY, PRECIPITATION AND OSMOLALITY
A clear solution of test item was obtained in DMSO up to a concentration of 100 mg/ml. There was no precipitation in the medium at any concentration tested.
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

CONTROLS
The number of aberrations found in the solvent controls was in the range of historical laboratory control data.
The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µl/ml) and cyclophosphamide (5 µg/ml) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Conclusions:

The test item is considered as being non-clastogenic under the tested conditions.

Executive summary:

The test item, dissolved in DMSO, was tested in a chromosome aberration assay in V79 cells in two independent experiments, in accordance with OECD guideline 473. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test (without and with metabolic activation using rodent S9 mix): experiment A with 3/20 h treatment/sampling time 250, 500, 1000 and 2000 μg/ml test item without S9 mix and 125, 250, 500 and 1000 μg/ml test item with S9 mix; experiment B with 20/20 h treatment/sampling time 62.5, 125, 250 and 500 μg/ml test item without S9 mix; experiment B with 20/28 h treatment/sampling time 62.5, 125, 250 and 500 μg/ml test item without S9 mix; experiment B with 3/28 h treatment/sampling time 125, 250, 500 and 1000 μg/ml test item with S9 mix.

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/ml) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts.

No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.

In the experiment B in the absence of metabolic activation one value at the dose of 62.5 µg/ml and in the presence of metabolic activation one value at the dose of 1000 µg/ml (5 aberrant cells excluding gaps/150 cells) were slightly above the 95 % control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells).

However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and thus, the findings were not considered as being biologically relevant.  

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µl/ml) and cyclophosphamide (5 µg/ml) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Conclusion

Test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic under the tested conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March from 06 to 22, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 29 July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures).
- Cells: Sub-line (K1) of Chinese hamster ovary cell line CHO. The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO K1 is a sub-line of CHO cell line.
- Methods for maintenance in cell culture if applicable: the cell stocks are kept in liquid nitrogen. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 ± C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks.
- Culturing: the CHO K1 cells for the study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/ml penicillin, 10 mg/ml streptomycin and 25 µg/ml amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).

MEDIA USED
- Periodically checked for Mycoplasma contamination: each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

MAIN EXPERIMENT: 125, 250, 500, 1000 and 2000 µg/ml, with and without metabolic activation.
PRELIMINARY EXPERIMENT for CYTOTOXICITY: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/ml, with and without metabolic activation.

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent/vehicle: the solvent is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test. The suitability was confirmed with the available laboratory’s historical database.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

PROCEDURES
- Preparation of the dishes: on the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item.
- Treatment: a 5-hour treatment in the presence and absence of S9-mix was performed.
- Number of cells: 5 x10^6 cells were each placed in sterile dishes.
- Incubation conditions: dishes were incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
- Post-exposure incubation: following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours.
- Count: after the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber.
- Precipitation check: solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment.
- Adjustement of cell number: in samples where sufficient cells survived, cell number was adjusted to 10^5 cells/ml. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

REPLICATES: duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix.

PLATING SURVIVAL
Following adjustment of the cultures to 2 × 10^5 cells/ml, samples from these cultures were diluted to 40 cells/ml.
A total of 5 ml (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm).
The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 4-6 days for growing colonies.
Then, colonies were fixed with methanol, stained with Giemsa and counted. Survivals were assessed by comparing the cloning efficiency of the test item treated groups to the negative (solvent) control.

EXPRESSION OF MUTANT PHENOTYPE: during the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2×10^6 cells were taken on days 1, 3, 6 and evaluated on day 8.

SELECTION OF THE MUTANT PHENOTYPE: at the end of the expression period, cultures from each dose level were adjusted to 2 × 10^5 cells / dish (4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/ml of thioguanine (6-TG).

PLATING OF VIABILITY: at the end of the expression period cell number in the samples was adjusted to 2 × 10^5 cells/ml. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.

FIXATION AND STAINING OF COLONIES: after the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination.

DETERMINATION OF CYTOTOXICITY
Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on cell toxicity. During the Pre-test on Toxicity (cytotoxicity assay), proliferating cells were trypsinised and cell suspensions was prepared in Ham's F12-10 medium. Cells were seeded into petri dishes (tissue cultures quality: TC sterile) at 2.5×10^6 cells each and incubated with culture medium.
After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence of S9 mix (50 ml/ml) and incubated at 37 °C for 5 hours.
After the treatment cells were washed and incubated in fresh Ham's F12-10 medium for 19 hours. 24 hours after the beginning of treatment, the cultures were washed with Ham's F12-5 medium and the cells were covered with trypsin-EDTA solution, counted and the cell concentration will be adjusted to 40 cells/ml with Ham's F12-10 medium. For each concentration of test solution or control solution, 5 ml will be plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 5-7 days for colony growing. Colonies were then be fixed with methanol and will be stained with Giemsa and the colonies will be counted. In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control.
Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments.

PRELIMINARY SOLUBILITY TEST
A non GLP Preliminary Solubility Test was performed suspending the test item in DMSO. A homogeneous suspension was obtained up to a concentration of 100 mg/ml.

METABOLIC ACTIVATION FRACTION
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

The S9 Mix (with Rat Liver S9)
Before adding to the culture medium the S9 Mix will be kept in an ice bath.
S9 mix preparation (concentration in the mix): HEPES 0.2 ml/ml, KCl 0.1 ml/ml, MgCl2 0.1 ml/ml, NADP 0.1 ml/ml, D-Glucose-6-phosphate (Monosodium salt) 0.1 ml/ml, F12 medium 0.1 ml/ml and S9 0.3 ml/ml.

ACCEPTANCE CRITERIA
The assay was considered valid as all the following criteria were met:
- The mutant frequency of concurrent negative controls is within the 95% control limits of the distribution of the laboratory’s historical negative control database.
- The positive control chemicals induced a statistically significant and biologically relevant increase in mutant frequency compared to the concurrent negative control. The increases are compatible with the laboratory historical positive control data base.
- Adequate number of cells and concentrations were analysable.
- Two experimental conditions with and without metabolic activation were tested.
- The highest concentration is adequate.
- The cloning efficiency of the negative controls is between the range of 60 % to 140 % on Day 1 and 70 % to 130 % on Day 8.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item 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,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95 % control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

Providing that all acceptability criteria are fulfilled, a test item 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 (based 95 % control limit).

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.

Species / strain:

Chinese hamster Ovary (CHO)

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:

There were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no significant differences between treatment and control groups and no dose-response relationships were noted.

CYTOTOXICITY
On Day 1, in the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 2000 µg/ml confirming the response seen in the dose selection cytotoxicity assays.

CONTROLS
All results were inside the distribution of the historical negative control data (based 95 % control limit) and no dose-related increase was observed in any of the cultures.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 µl/ml) and 7,12-dimethyl benz[a]anthracene (20 µg/ml). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. Thus, the study is considered valid.

OSMOLALITY AND pH
The osmolality and pH values of test item solutions did not show any significant alterations compared to the concurrent control groups in the pre-test on toxicity and main mutation assay.

SOLUBILITY OF TEST ITEM
A homogeny suspension was obtained up to a concentration of 100 mg/ml. For examined test item concentrations no precipitation in the medium was noted.

Preliminary experiment for cytotoxicity (5 hours treatment)

Group and dose (µg/ml)	S9-mix	Treatment/time/hrs	Number of colonies/200cells/dish			Mean	Relative*survival (%)
			dish 1	dish 2	dish 3
Untreated Control	–	5	203	204	205	204.0	101
Solvent Control (DMSO)	–	5	202	202	203	202.3	100
Test item 15.6	–	5	203	199	201	201.0	99
Test item 31.3	–	5	202	198	204	201.3	100
Test item 62.5	–	5	203	201	202	202.0	100
Test item 125	–	5	200	202	203	201.7	100
Test item 250	–	5	201	200	198	199.7	99
Test item 500	–	5	204	198	204	202.0	100
Test item 1000	–	5	200	200	203	201.0	99
Test item 2000	–	5	201	202	200	201.0	99
Untreated Control	+	5	200	203	203	202.0	100
Solvent Control (DMSO)	+	5	201	202	202	201.7	100
Test item 15.6	+	5	203	198	203	201.3	100
Test item 31.3	+	5	199	200	201	200.0	99
Test item 62.5	+	5	203	204	201	202.7	100
Test item 125	+	5	200	203	202	201.7	100
Test item 250	+	5	201	203	199	201.0	100
Test item 500	+	5	195	190	194	193.0	96
Test item 1000	+	5	192	191	188	190.3	94
Test item 2000	+	5	174	176	180	176.7	88

* Relative to Solvent Control

Conclusions:

The test item is considered as being non-mutagenic in the system.

Executive summary:

The substance was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation: 125, 250, 500, 1000 and 2000 µg/ml, with and without S9-mix.

In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 µg/ml, based on the updated OECD guideline 476 (2016). Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted. All results were inside the distribution of the historical negative control data (based 95 % control limit).

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulfonate and 7, 12 -dimethyl benzanthracene caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, the substance tested up to the maximum recommended concentration with and without metabolic activation system over a 5 hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).

Conclusion

The test item is considered as being non-mutagenic in the system.

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 gene mutation assay in bacteria, in vitro gene mutation on mammalian cells and in vitro chromosomal aberration assay.

IN VITRO BACTERIA GENE MUTATION ASSAY

The substance was tested for mutagenicity with strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2 uvrA. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 µg/plate to 5000 µg/plate was used. The test compound proved to be not toxic to the bacteria. 5000 µg/plate was chosen as top dose level for the mutagenicity study. In the absence of the metabolic activation system the test compound did not show a dose dependent influence on the number of revertants in any of the bacterial strains. Also in the presence of metabolic activation system, treatment of the cells with test item did not result in relevant increases in the number of revertant colonies.

IN VITRO MAMMALIAN CELL GENE MUTATION ASSAY

The substance was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation: 125, 250, 500, 1000 and 2000 µg/ml, with and without S9-mix.

In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for soluble, low cytotoxic substances is 2000 µg/ml, based on the updated OECD guideline 476 (2016). Phenotypic expression was evaluated up to 8 days following exposure. In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted. All results were inside the distribution of the historical negative control data (based 95 % control limit).

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulfonate and 7, 12 -dimethyl benzanthracene caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, the substance tested up to the maximum recommended concentration with and without metabolic activation system over a 5 hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).

IN VITRO CHROMOSOMAL ABERRATION

The test item, dissolved in DMSO, was tested in a chromosome aberration assay in V79 cells in two independent experiments, in accordance with OECD guideline 473. The concentrations were selected on the basis of a pre-test: Experiment A with 3/20 h treatment/sampling time 250 - 2000 μg/ml without S9 mix and 125 - 1000 μg/ml with S9 mix; Experiment B with 20/20 h and 20/28 h treatment/sampling time 62.5 - 500 μg/ml without S9 mix; Experiment B with 3/28 h treatment/sampling time 125 - 1000 μg/ml with S9 mix

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts.

No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.

In the experiment B in the absence of metabolic activation one value at the dose of 62.5 µg/ml and in the presence of metabolic activation one value at the dose of 1000 µg/ml (5 aberrant cells excluding gaps/150 cells) were slightly above the 95 % control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells).

However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and thus, the findings were not considered as being biologically relevant.  

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent and positive controls were in the range of the historical laboratory data, thus, the study is considered valid.

Therefore, it was concluded that the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic under the tested conditions.

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

- 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).