Reaction mass of trisodium [m[7-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-8-hydroxynaphthalene-1,6-disulphonato(7-)]]dicuprate(3-) and trisodium [m[4-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-3-hydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(3-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Remarks:

source of read across study record

Adequacy of study:

key study

Study period:

From November 28th to December 15tth, 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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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:

other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Method

Species / strain

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
- Type and identity of media including CO2 concentration if applicable:
- 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.

Controls

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 x10^6 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 2x10^6 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 x 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. l

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

Results and discussion

Test results

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 OSMOLARITY
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.