[No public or meaningful name is available]

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From April 25 to May 11, 1990

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:

Details for read across approach are included into the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Regular checking: regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory according to Ames et al.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. The nutrient medium contains per litre: 8 g Difco Nutrient Broth, 5 g NaCl. The bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal activation system

Test concentrations with justification for top dose:

10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate

Vehicle / solvent:

- Vehicle: aqua dest, for test item and sodium azide. DMSO for 4-NOPD and 2-AA.
- Justification for choice of vehicle: the vehicle was chosen because of its solubility.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation, two independent experiments.

AGAR
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121 °C in an autoclave.
- Overlay Agar: the overlay agar contains per litre 6.0 g Difco Bacto Agar, 6.0 g NaCl, 10.5 mg L-histidine × HCl x H2O and 12.2 mg biotin. Sterilizations were performed at 121 °C in an autoclave.

NUMBER OF REPLICATIONS: each concentration, including the controls, was tested in triplicate.

EXPERIMENTAL PERFORMANCE
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control);
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation);
100 µl bacteria suspension (cf. test system, pre-culture of the strains);
2000 µl overlay agar
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates.
The experimental conditions in this pre-experiment were the same as described for the experiment.
Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 preparation
The S9 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male Wistar rats, strain WU (weight ca. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in RC1 was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70 °C. Small numbers of the ampoules are kept at -20 “C for only several weeks before use.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.

S9 mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Evaluation criteria:

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows: a test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

CYTOTOXICITY
Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 1535 at 5000.0 µg/plate (exp. I; without S9 mix), in TA 1537 at 1000.0 and 5000.0 µg/plate (exp. I; without S9 mix), and in TA 98 at 1000.0 µg/plate (exp.II; with S9 mix), and 5000.0 µg/plate (exp.I, and II; with S9 mix).
The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

CONTROLS
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusions:

The test item is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate.

Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 1535 at 5000.0 µg/plate (exp. I; without S9 mix), in TA 1537 at 1000.0 and 5000.0 µg/plate (exp. I; without S9 mix), and in TA 98 at 1000.0 µg/plate (exp.II; with S9 mix), and 5000.0 µg/plate (exp.I, and II; with S9 mix).

The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, test item is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 2

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 May 04 to 19, 1990

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:

Details for read across approach are included into the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Regular checking: regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory according to Ames et al.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. The nutrient medium contains per litre: 8 g Difco Nutrient Broth, 5 g NaCl. The bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

Hamster liver microsomal activation system

Test concentrations with justification for top dose:

10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate

Vehicle / solvent:

- Vehicle: aqua dest, for test item and sodium azide. DMSO for 4-NOPD and 2-AA.
- Justification for choice of vehicle: the vehicle was chosen because of its solubility.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation; two independent experiments.

AGAR
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121 °C in an autoclave.
- Overlay Agar: the overlay agar contains per litre 6.0 g Difco Bacto Agar, 6.0 g NaCl, 10.5 mg L-histidine × HCl × H2O and 12.2 mg biotin. Sterilizations were performed at 121 °C in an autoclave.

NUMBER OF REPLICATIONS: each concentration, including the controls, was tested in triplicate.

EXPERIMENTAL PERFORMANCE
The following materials were mixed in a test tube and gently shaken at 30 °C for 30 min:
100 µl test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control);
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation);
100 µl bacteria suspension (cf. test system, pre-culture of the strains).
After preincubation 2.0 ml of molten 45 °C overlay agar was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates.
The experimental conditions in this pre-experiment were the same as described for the experiment.
Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 preparation
The S9 liver microsomal fraction was obtained from the liver of 7 - 8 weeks old male Syrian golden hamster.
After cervical dislocation the livers of the animals were removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in bidistilled water and homogenised. The homogenate, diluted 1:3 in sodium phosphate buffer was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70 °C. Small numbers of the ampoules are kept at -20 “C for only several weeks before use.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.

S9 mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 20 mM glucose-6-phosphate, 2.8 unites/ml glucose-6-phosphate dehydrohenase, 4 mM NADP, 2.0 mM NADH, 2.0 mM FMN in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al and Prival and Mitchell.

Evaluation criteria:

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows: a test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA 98 at 5000.0 µg/plate (exp. II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

CYTOTOXICITY
Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 98 at 5000.0 µg/plate in the presence of metabolic activation in experiment II.

CONTROLS
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusions:

The test item is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations according to the pre-incubation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate.

Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 98 at 5000.0 µg/plate in the presence of metabolic activation in experiment II.

The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, test item is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 28 to December 15, 2017

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

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: the CHO cell line was originally derived from the ovary of a female Chinese hamster. The CHO K1 is a sub-line of CHO cell line. The CHO K1 cell line was purchased.
- Supplier: ECACC (European Collection of Cell Cultures)
- Storage: the cell stocks are kept in liquid nitrogen.

MEDIA USED
- Periodically checked for Mycoplasma contamination: each batch of cells was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary; results were fully documented within the raw data file.
- Preparation of cells: 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.

GROWING CONDITIONS
The CHO K1 cells for the study were grown in Ham's F12 medium 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 %).
During the 5 treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 %. The selection medium for TG resistant mutants contained 3.4 µg/ml 6-thioguanine (6-TG).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 Mix

Test concentrations with justification for top dose:

MAIN EXPERIMENT: 125, 250, 375, 500 and 750 µg/ml without S9-mix; 31.3, 62.5, 125, 250, 500 and 750 µg/ml with S9.

Vehicle / solvent:

- Vehicle: the test item was dissolved in Ham's F12 medium and diluted prior to treatment.
- Justification for choice of vehicle: the vehicle resulted to be compatible with the survival of the CHO cells.
- Preparation: test item was prepared in a concentration of 25 mg/ml in Ham's F12 medium (stock solution) at the first step. Correction of concentration for active component content of 88.3 % was made with correction factor 1.132. The necessary amount of test item was weighed into a calibrated volumetric flask. A partial volume of vehicle was added and the formulation was stirred until homogeneity is reached. The formulation was diluted by serial dilutions to obtain the dosing formulations for lower doses. The appropriate amount of these dosing formulations were diluted with Ham's F12 medium or Ham's F12 medium + S9 mix to obtain the test concentrations. All dose formulations were prepared directly prior to the treatment of the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

vehicle

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

CULTURE MEDIUM: on the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item.

DURATION
- Preincubation period: 5 × 10^6 cells were each placed in sterile dishes and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
- Exposure duration: 5-hour treatment. Following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours.

NUMBER OF REPLICATIONS: 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 FOR SURVIVAL
Following adjustment of the cultures to 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 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.

ERXPRESSION OF THE 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 FOR 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 FOR 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.

CELLS 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.

DETERMINATION OF CYTOTOXICITY
A GLP pre-test on toxicity was performed.
During the pre-test on toxicity (cytotoxicity assay), the cultures (more than 50 % confluent) was trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells was seeded into petri dishes (tissue cultures quality: TC sterile) at 5x10^6 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 µl/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 was adjusted to 40 cells/ml with Ham's F12-10 medium. For each concentration of test solution or control solution, 5 ml was 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 6 days for colony growing. Colonies were then fixed with methanol and was stained with Giemsa and the colonies were 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. In addition, pH and osmolality was considered for dose level selection.

MAMMALIAN MICROSOMAL FRACTION S9
- Supplier: the S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH.
- Composition: N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid 0.2 ml/ml, KCl 0.1 ml/ml, MgCl2 0.1 ml/ml, β-Nicotinamide Adenine Dinucleotide Phosphate 0.1 ml/ml, D-Glucose 6 phosphate (Monosodium salt) 0.1 ml/ml, Ham’s F12 medium 0.1 ml/ml, S9 0.3 ml/ml.
- Storage: before adding to the culture medium the S9 mix was kept in an ice bath.

ASSAY 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 were fulfilled, a test chemical is considered clearly negative because:
- 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

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

On day 1, there was very clear evidence of toxicity at the highest tested concentration with the test item in presence and absence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
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. All values were within the range of the laboratory historical solvent control data and no dose-related increase was observed in any of the cultures.

CONTROLS
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.

pH AND OSMOLALITY
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 AND CONCENTRATION SELECTION
A clear solution was obtained up to a concentration of 25 mg/ml. For examined test item concentrations, no precipitation in the medium was noted.

PRE-TEST ON CYTOTOXICITY

Test group	Dose µg/ml	S9-mix	Treatment/time/ hour	Number of colonies/200cells/dish			Mean	Relative^asurvival in percent
				dish 1	dish 2	dish 3
Solvent control (Ham's F12 medium)		–	5	201	203	202	202.0	100
Direct Blue 267	15.6	–	5	201	203	201	201.7	100
	31.3	–	5	201	204	200	201.7	100
	62.5	–	5	201	197	198	198.7	98
	125	–	5	193	198	194	195.0	97
	250	–	5	166	169	168	167.7	83
	500	–	5	90	88	86	88.0	44
	1000	–	5	0	0	0	0.0	0
	2000	–	5	0	0	0	0.0	0
Solvent control (DMO)	–	+	5	198	200	199	199.0	100
Direct Blue 267	15.6	+	5	190	195	194	193.0	97
	31.3	+	5	188	186	190	188.0	94
	62.5	+	5	134	132	131	132.3	66
	125	+	5	120	122	120	120.7	61
	250	+	5	105	106	102	104.3	52
	500	+	5	80	81	79	80.0	40
	1000	+	5	0	0	0	0.0	0
	2000	+	5	0	0	0	0.0	0

Conclusions:

The test item is considered as being non-mutagenic, under tested conditions.

Executive summary:

The test item, was assessed in a mammalian gene mutation test in CHO-K1 cells. The test item was dissolved in Ham's F12 medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver.

Mutation assay was performed at the following concentrations and treatment intervals: at 125, 250, 375, 500 and 750 µg/ml test item without S9-mix; at 31.3, 62.5, 125, 250, 500 and 750 µg/ml test item with S9.

In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity of test item. 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.

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 (1.0 µl/ml) and 7, 12 -dimethyl benzanthracene (20 µg/ml) 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 cytotoxic concentrations 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, under tested conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Non genotoxic

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 22 to November 27, 1990

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:

Details for read across approach are included into the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, Basel, Switzerland.
- Number of animals: 84 (42 males and 42 females).
- Age at start of acclimatization: minimum 10 weeks.
- Weight at study initiation: approximately 30 g.
- Health conditions: according to the suppliers assurance, the animals were in healthy condition. The animals were under quarantine in the animal house of testing laboratory for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.
- Assigned to test groups randomly: the animals were distributed into the test groups at random and identified by cage number.
- Fasting period before study: approximately 18 hours before treatment with the test article the animals received no food but water ad libitum.
- Housing: single; Makrolon Type I cages, with wire mesh top. Granulated soft wood bedding.
- Diet: pelleted standard diet.
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 21± 3 °C
- Relative humidity: 30 - 70 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

- Vehicle: aqua dest.
- Justification for choice of vehicle: the vehicle was chosen to its nontoxicity for the animals.

Details on exposure:

DOSE VOLUME
At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The volume administered was 20 ml/kg bw.

PREPARATION OF DOSING SOLUTIONS
On day of the experiment, the test article was dissolved in aqua dest.

Frequency of treatment:

The animals received the test article once.

Post exposure period:

Sampling of the bone marrow was collected at 24, 48 and 72 hours.

Dose / conc.:

400 mg/kg bw/day

No. of animals per sex per dose:

Six males and six females were assigned to each test group.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: cyclophosphamide.
- Vehicle: physiological saline.
- Route of administration: orally, singly.
- Doses / concentrations: 30 mg/kg bw.

Tissues and cell types examined:

Bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: a preliminary study (4 pre-experiments) on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study.

TREATMENT AND SAMPLING TIMES: sampling of the bone marrow was collected at 24, 48 and 72 hours.

DETAILS OF SLIDE PREPARATION: the animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grfinwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: evaluation of the slides was performed using NIKON microscopes with 100× oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.
A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at anyone of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean number of normochromatic erythrocytes was not substantially after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls indicating that test item had no cytotoxic properties.
In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

POSITIVE CONTROL
Cyclophosphamide administration showed a distinct increase of induced micronuleus frequency.

PRE-EXPERIMENT FOR TOXICITY
First pre-experiment: in the pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w. test item dissolved in aqua dest.. The volume administered was 20 ml/kg b.w.
The treated animals expressed toxic reactions: reduction of spontaneous activity, abdominal position, eyelid closure, apathy. All animals died within 6 hours after treatment.

Second pre-experiment: in the pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. test item dissolved in aqua dest.. The volume administered was 20 ml/kg b.w.
All treated animals expressed toxic reactions: reduction of spontaneous reaction one male and two females; abdominal position two females; eyelid closure two females; apathy one male and two females; lethalities one male and two females.

Third pre-experiment: in the pre-experiment 4 animals (2 males, 2 females) per dose group received orally a single dose of 400, or 500 mg/kg b.w. test item, respectively, dissolved in aqua dest. The volume administered was 10 ml/kg b.w.
400 mg/kg b.w.: reduction of spontaneous reaction two males and two females; eyelid closure one male and one female.
500 mg/kg b.w.: reduction of spontaneous reaction two males and two females; eyelid closure in one male and one female; apathy one male and one female; lethalities one male.

Fourth pre-experiment: in the pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 300 mg/kg b.w. test item dissolved in aqua dest.. The volume administered was 10 ml/kg b.w.
The animals expressed no toxic reactions.
On the basis of these results 400 mg/kg b.w. test item were estimated to be the maximum tolerated dose.

Summary of results

Test group	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei	Range	PCE/NCE
Solvent	0	24	0.06 %	0 - 3	1000 / 880
Test item	400	24	0.06 %	0 - 3	1000 / 935
Cyclophosphamide	30	24	1.44 %	7 - 30	1000 / 899
Solvent	0	48	0.06 %	0 - 2	1000 / 917
Test item	400	48	0.09 %	0 - 3	1000 / 973
Solvent	0	72	0.07 %	0 - 3	1000 / 765
Test item	400	72	0.06 %	0 - 4	1000 / 911

Conclusions:

The test item is considered to be non-mutagenic in the micronucleus assay.

Executive summary:

The study was performed to investigate the potential of test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was dissolved in aqua dest.; the solvent was used as negative control. The volume administered orally was 20 ml/kg b.w. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval, 400 mg/kg b.w.

In pre-experiments this dose level was estimated to be the maximum tolerated dose.

After treatment with the test article the ratio between PCEs and NCEs was not substantially affected when compared to the corresponding negative controls thus indicating no cytotoxic effects.

In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

Conclusion

It can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, test item is considered to be non-mutagenic in the micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Evaluation of the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro gene mutation on mammalian cells conducted on Direct Blue 267 and in vitro AMES tests and in vivo chromosomal aberration performed on the structural analogue Similar Substance 01.

The read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included into the IUCLID section 13).

BACTERIA GENE MUTATION ASSAY

Two AMES tests are available for Similar Substance 01; both tests were performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. In one case the classical protocol for AMES test was followed; in the other one, the procedure described by Prival and Mitchell was followed.

In the classical AMES test the plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain. The presence of liver microsomal activation did not influence these findings (Poth, 1990).

In the Prival test, toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 98 at 5000.0 µg/plate in the presence of metabolic activation in experiment II. The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain (Poth, 1990).

 

MAMMALIAN CELL GENE MUTATION ASSAY

The test item, was assessed in a mammalian gene mutation test in CHO-K1 cells. Mutation assay was performed at the following concentrations and treatment intervals: at 125, 250, 375, 500 and 750 µg/ml test item without S9-mix; at 31.3, 62.5, 125, 250, 500 and 750 µg/ml test item with S9.

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. 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; thus, the study was considered valid. In conclusion, the substance, tested up to the cytotoxic concentrations 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 (Béres, 2018).

 

CHROMOSOMAL ABERRATION ASSAY

The chromosomal aberration potential has been investigated taken into account the available information on the structural analogue, Similar Substance 01.

The study was performed to investigate the potential of Similar Substance 01 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. A dose level of 400 mg/kg bw of the test article was investigated at 24 h, 48 h, and 72 h preparation interval. After treatment with the test article, the ratio between PCEs and NCEs was not substantially affected when compared to the corresponding negative controls thus indicating no cytotoxic effects (Völkner, 1990).

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.

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).