1,3'-Bipyridinium, 5'-[[4-[[4-[(1',2'-dihydro-6'-hydroxy-3,4'-dimethyl-2'-oxo[1,3'-bipyridinium]-5'-yl)azo]benzoyl]amino]phenyl]azo]-1',2'-dihydro-6'-hydroxy-3,4'-dimethyl-2'-oxo-, salt with 2-hydroxypropanoic acid (1:2)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 30th to February 15th, 2018

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: 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 ca. 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: 250, 500, 1000 and 2000 µg/ml, with and without metabolic activation.
PRELIMINARY EXPERIMENT for CYTOTOXICITY: 125, 250, 500, 1000, 1500 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 × 10^6 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 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 × 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.

MEASUREMENT PF pH AND OSMOLALITY
The pH and osmolality of the negative (solvent) control and test item solutions were determined in Pre-test on Toxicity (Concentration selection) and the Main Mutation Assay.

DETERMINATION OF CYTOTOXICITY
Treatment concentrations for the mutation assay were selected on the basis of the result of Pre-test on Toxicity. 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 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 µld 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. The results of pre-test on cell toxicity were used for dose selection of the test item concentrations used in the Main Mutation Assay.

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 protein concentrations of the S9 batch used in the experiments were 33.7, 33.8, 35.7 and 37.8 mg/ml.

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 database.
- 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:

In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control at any concentration tested in the absence and presence of metabolic activation. In the absence and in the presence of S9 mix, in the cultures at any concentrations tested the mean mutation frequency a little bit exceeded the 95 % confidence interval of the historical control data. Statistically significant differences to the historical control data was observed at concentration 2000 µg/ml in 1 out of 4 cultures. These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls were observed.

CYTOTOXICITY
On Day 1, there was no cytotoxicity observed at the tested concentrations with the test item in absence of metabolic activation (S9 mix) and low cytotoxicity observed at the tested concentrations with the test item in presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays.

OSMOLALITY AND pH
The osmolality 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. The pH values of test item solutions showed a dose associated decrease in the acceptable range compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay.

SOLUBILITY
A clear solution was obtained up to a concentration of 100 mg/ml. For examined test item concentrations no precipitation in the medium was noted.

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.

Preliminary experiment for cytotoxicity (5 hours treatment)

Test group	S9-mix	Treatment/time/ hour	Number of colonies/200cells/dish			Mean	Relative^asurvival (%)
Untreated Control	–	-	206	201	203	203.3	101
Solvent Control (DMSO)	–	5	202	200	204	202.0	100
Test item, 125µg/ml	–	5	203	199	203	201.7	100
Test item, 250µg/ml	–	5	205	202	203	203.3	101
Test item, 500µg/ml	–	5	199	203	204	202.0	100
Test item, 1000µg/ml	–	5	203	202	204	203.0	100
Test item, 1500µg/ml	–	5	194	200	197	197.0	98
Test item, 2000µg/ml	–	5	194	199	198	197.0	98
Untreated Control	+	-	201	201	203	201.7	103
Solvent Control (DMO)	+	5	195	196	196	195.7	100
Test item, 125µg/ml	+	5	191	190	195	192.0	98
Test item, 250µg/ml	+	5	194	192	192	192.7	98
Test item, 500µg/ml	+	5	152	158	157	155.7	80
Test item, 1000µg/ml	+	5	135	139	140	138.0	71
Test item, 1500µg/ml	+	5	128	132	127	129.0	66
Test item, 2000µg/ml	+	5	124	126	122	124.0	63

* Relative to Solvent Control

Conclusions:

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).
Thus, the test item is considered as being non-mutagenic in this system.

Executive summary:

The test item 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 using S9 mix of phenobarbital and β-naphthoflavone induced rat liver. Mutation Assay was performed at the following concentrations and treatment intervals: mutation assay 5-hour treatment period with and without S9-mix at 250, 500, 1000 and 2000 µg/ml.

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 statistically significant increases in mutation frequency when compared to the concurrent solvent control at any concentration tested in the absence and presence of metabolic activation. In the absence and in the presence of S9 mix, in the cultures at any concentrations tested the mean mutation frequency a little bit exceeded the 95 % confidence interval of the historical control data. Statistically significant differences to the historical control data was observed at concentration 2000 µg/ml in 1 out of 4 cultures. These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls were observed.

The osmolality 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. The pH values of test item solutions showed a dose associated decrease in the acceptable range compared to the concurrent control groups.

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls 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.

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).

Thus, the test item is considered as being non-mutagenic in this system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Principles of method if other than guideline:

Escherichia coli strain WP2 uvrA- were tested in order to assess the test item mutagenicity activity.
A first experiments has been performed to evaluate the antibacterial activity potential at the concentration of 5000 µg/plate; after that, the mutagenicity assay has been performed at concentrations between 5000 - 10 µg/plate.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Escherichia coli strain WP2 uvrA- was kept at 4 °C in deep soft nutrient agar.
The day before the test, a fresh culture was prepared in nutrient broth and incubated overnight at 37 °C.
Concurrently with each series of test, the above cultre was checked for relevant characters, namely:
- tryptophan requirement
- uvrA character
- number of spontaneous tryptophan prototrophe revertants
- number revertants induced by methylmethane sulphonate (a known mutagen of the base pair-substitution type).

Metabolic activation:

with and without

Metabolic activation system:

liver supernatant fraction (rats)

Test concentrations with justification for top dose:

ANTIBACTERIAL ACTIVITY: 5000 µg/plate
MUTAGENESIS TEST: 5000, 1000, 500, 100, 50, 10 µg/plate

Vehicle / solvent:

Test item was dissolved 50 mg/ml in dimethylsulfoxide (DMSO) and further diluted in the same solvent if needed.
It was determined that the amounts of solvent added to the plates did not interfere with the results of mutagenesis or antibacterial test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml of the solvent mixture

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Details on test system and experimental conditions:

MEDIA
Semi-enriched medium (SEM) had the following composition: K2HPO4 7 g/l, KH2PO4 3 g/l, Sodio citrato 0.5 g/l, MgSO4 × 7H2O 0.1 g/l, (NH4)2SO4 1 g/l, Nutrient broth 0.3 g/l, Glucose 2 g/l, Agar 15 g/l.
It was prepared by adding 750 ml of 2 % molten autoclave sterilized water agar with 250 ml of 4 times con centrated salt solution, 10 ml of 20 % glucose solution and 12.5 ml of Oxoid N. 2 nutrient broth autoclaved separately. 10 cm plastic petri dishes were prepared cogtaining 20 ml each of SEM agar and used within 3 days from preparation.
Top agar (0.6 % agar in distilled water) was dispersed in 2 ml amounts into 16 × 100 mm glass tube kept at 42 °C.

ANTIBACTERIAL ACTIVITY TEST
For testing the antibacterial activity 0.1 ml of solvent containing the required amount of substance, 0.1 ml of a 10^-5 dilution of an overnight culture of WP2 uvrA containing between 100 and 500 colony forming units (CFU) and, if requested, 0.5 ml of S9-mix were added in sequence to each top agar tube.
The contents of each tube were quickly mixed and poured on the surface of a histidine containing MM plate.

MUTAGENESIS TEST
In view of the results of antibacterial activity, the substance was tested for mutagenic activity at the doses of 5000 to 10 µg/plate.
0.1 ml of solvent containing the required amounts of substance, 0.1 ml of overnight culture of WP2 uvrA containing 10^7 bacteria and, if required, 0.5 ml of S9-mix were added in sequence to each top agar tube. The contents of each tube were quickly mixed and poured on the surface of a SEM plate.

NUMBER OF REPLICATIONS
Tests were carried out in triplicate.

CELLS COUNT
Antibacterial activity: colonies were counted after 24 hours of incubation at 37 °C.
Mutagenesis test: revertant colonies were counted after 2 days of incubation at 37 °C.

S9-MIX
A liver supernatant fraction was prepared from Wistar male rats (170-250 g). Three rats each received a single i.p. injection of Aroclor 1254 (200 mglml in corn oil, 500 mg/kg). Five days after injection the rats were killed by cervical dislocation. The livers washed with 0.15 KCl ice cold solution were weighted and transferred to a beaker containing 3 ml of KCl 0.15 M solution per gram of net weight of liver, minced with steril scissors and homogenized in a Potter-Evenhjem apparatus with a teflon pestle.
The homogenate was centrifuged for 10 min at 9000 g and the supernatant decanted and stored at -80 °C.
100 ml of S9-mix were prepared by mixing: 10 ml of liver homogenate, 4 ml of nicotinamide adenine dinucleotide phosphate (NADP) 0.1 M, 0.5 ml of glucose-6-phosphate (G6P) 1 M, 2 ml of KCl 1.65 M, 2 ml of MgCl2 0.4 M, 50 ml of phosphate buffer pH 7.4 0.2 M, 31.5 ml of sterile distilled water.
NADP and G6P stock solutions were millipore filter sterilized and stored at -20 °C. The stock salt solutions were autoclave sterilized and stored at 4 °C. Each batch of S9-mix ·was tested for sterility and activity.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no antibacterial activity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENESIS TEST
None of the tested doses of test item increases the number of revertants over the spontaneous levels. It may be concluded that the substance is without any mutagenic activity on the bacterial strain used in the test.

ANTIBACTERIAL ACTIVITY
Result show that test item is without antibacterial activity up tn the dose of 5000 µg/plate: counts of CFUs in the control plates and in plates containing the highest dose of compound did not differ significantly.

Mutagenicity

Strain	Dose µg/plate	Revertant colonies + S9-mix		Revertant colonies - S9-mix
		Mean	R*	Mean	R*
WP2 uvrA-	5000	21	< 2	19	< 2
	1000	22	< 2	18	< 2
	500	24	< 2	16	< 2
	100	24	< 2	20	< 2
	50	22	< 2	18	< 2
	10	17	< 2	17	< 2
	0	22		17

* times of increase over the spontaneous reversion rate. Values less than 2 were considered not significant of mutagenic activity.

Antibacterial activity

Strain	Dose µg/plate	Colony counts + S9-mix					Colony counts - S9-mix
		CFU/plate			Mean	% survival	CFU/plate			Mean	% survival
WP2 uvrA^-	0	184	196	220	200	99	190	201	214	202	100
	5000	187	189	215	197		201	207	220	209

CFU: colony forming units

Control of E. coli WP2 uvrA^-

Grown in MM	-
Grown in MM + tryptophan	+
uvrA character	highly UV sensitive
Spontaneous reversion rate*	9, 11, 12
Reversion by 1 mg of MMS	250, 318, 356

MM: minimal medium

MMS: methylmethanesulphonate

*Number of revertant colonies per plate

Control of S9 -mix

Sterility	OK
Activity	Number of revertants/plate
WP2 uvrA^-	9, 11, 12
WP2 uvrA^- + S9-mix	20, 25, 26
WP2 uvrA^- + DMNA (5 µg)	8, 10, 14
WP2 uvrA^-+ DMNA (5 µg) + S9-mix	352, 411, 428

DMNA = dimethylnitrosamine by liquid test.

Conclusions:

The substance did not showed any mutagenic activity on the bacterial strain used in the test, with and without metabolic activation.

Executive summary:

Escherichia coli strain WP2 uvrA- were tested in order to assess the test item mutagenicity activity.

A first experiments has been performed to evaluate the antibacterial activity potential at the concentration of 5000 µg/plate; after that, the mutagenicity assay has been performed at concentrations between 5000 - 10 µg/plate.

Result show that test item is without antibacterial activity up to the dose of 5000 µg/plate: counts of CFUs in the control plates and in plates containing the highest dose of compound did not differ significantly.

None of the tested doses of test item increases the number of revertants over the spontaneous levels.

Conclusion

The substance did not showed any mutagenic activity on the bacterial strain used in the test, with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Principles of method if other than guideline:

Ames strains of Salmonella typhimurium TA1535, TA1537, TA1538, TA100 and TA98 were tested in order to assess the test item mutagenicity activity. A first experiment has been performed on TA1535 and TA98 to evaluate the antibacterial activity potential of the substance; then, the mutagenicity assay has been performed on all strains.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98

Details on mammalian cell type (if applicable):

Ames strains of Salmonella typhimurium TA1535, TA1537, TA1538, were kept at 4 °C in deep soft nutrient agar. Strains TA100 and TA98 were kept at -80 °C in nutrient broth plus ampicillin (20 µg/ml) and dimethylsufoxide (80 µl/ml).

The day before the test, fresh cultures were prepared by inoculating 10 ml of nutrient broth with each strain, which were incubated overnight at 37 °C. Concurrently with each series of test, cultures were checked for relevant characters, namely:
- histidine requirement
- biotin requirement
- uvrB character
- rfa character
- presence or absence of the pKM101 plasmid

Metabolic activation:

with and without

Metabolic activation system:

liver supernatant fraction (rats)

Test concentrations with justification for top dose:

ANTIBACTERIAL ACTIVITY: 5000 µg/plate
MUTAGENESIS TEST: 5000, 1000, 500, 100, 50, 10 µg/plate

Vehicle / solvent:

Test item was dissolved 50 mg/ml in dimenthylsulfoxide (DMSO) and further diluted in the same solvent according the need.
It was determined that the amounts of solvent added to the plates did not interfere with the results of mutagenesis or antibacterial test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml of the solvent mixture

Positive controls:

yes

Positive control substance:

9-aminoacridine

ethylmethanesulphonate

methylmethanesulfonate

other: 2-aminofluorene 10 µ + S9-mix

Details on test system and experimental conditions:

MEDIA
Vogel-Bonner E agar medium (VB) has the following composition: MgSO4 × 7H2O 0.2 g/l, Citric acid × H2O 2 g/l, K2HPO4 10 g/l, NaNH4HPO4 × 4H2O 3.5 g/l, Glucose 2 g/l, Agar 15 g/l
It was prepared by mixing 750 ml of 2 % molten autoclave sterilized water agar with 250 ml of 4 times concentrated salt solution and 10 ml of 20 % glucose solution autoclaved separately. For the antibacterial test histidine was also added to give a final concentration of 10 µg/ml. 10 em plastic petri dished were prepared containing 20 ml each of VB agar and used within 3 days from preparation. Top agar was prepared by mixing 100 ml of autoclave sterilized 0.6 % agar and 0.5 % NaCl in water with 10 ml of a millipore filter sterilized 0.5 mM histidine, 0.5 mM biotin solution. Top agar was dispensed in 2 ml amounts into 16×100 mm glass tubes kept at 42 °C.

ANTIBACTERIAL ACTIVITY TEST
For testing the antibacterial activity 0.1 ml of solvent containing the required amount of substance, 0.1 ml of a 10^-5 dilution of an overnight culture of TA1535 or TA98 containing between 100 and 500 colony forming units (CFU) and, if requested, 0.5 ml of S9-mix were added in sequence to each top agar tube.
The contents of each tube were quickly mixed and poured on the surface of a histidine containing VB plate.

MUTAGENESIS TEST
In view of the results of antibacterial activity, the substance was tested for mutagenic activity at the doses of 5000 to 10 µg/plate.
0.1 ml of solvent containing the required amounts of substance, 0.1 ml of overnight culture of each strain containing 10^7 bacteria and, if required, 0.5 ml of S9-mix were added in sequence to each top agar tube. The contents of each tube were quickly mixed and poured on the surface of a VB plate.

NUMBER OF REPLICATIONS
Tests were carried out in triplicate.

CELLS COUNT
Antibacterial activity: colonies were counted after 24 hours of incubation at 37°C.
Mutagenesis test: revertant colonies were counted after 2 days of incubation at 37 °C.

S9-MIX
A liver supernatant fraction was prepared from Wistar male rats (170-250 g). Three rats each received a single i.p. injection of Aroclor 1254 (200 mglml in corn oil, 500 mg/kg). Five days after injection the rats were killed by cervical dislocation. The livers washed with 0.15 KCl ice cold solution were weighted and transferred to a beaker containing 3 ml of KCl 0.15 M solution per gram of net weight of liver, minced with steril scissors and homogenized in a Potter-Evenhjem apparatus with a teflon pestle.
The homogenate was centrifuged for 10 min at 9000 g and the supernatant decanted and stored at -80 °C.
100 ml of S9-mix were prepared by mixing: 10 ml of liver homogenate, 4 ml of nicotinamide adenine dinucleotide phosphate (NADP) 0.1 M, 0.5 ml of glucose-6-phosphate (G6P) 1 M, 2 ml of KCl 1.65 M, 2 ml of MgCl2 0.4 M, 50 ml of phosphate buffer pH 7.4 0.2 M, 31.5 ml of sterile distilled water.
NADP and G6P stock solutions were millipore filter sterilized and stored at -20 °C. The stock salt solutions were autoclave sterilized and stored at 4 °C. Each batch of S9-mix ·was tested for sterility and activity.

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no antibacterial activity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENESIS TEST
None of the tested doses of test item increases the number of revertants over the spontaneous levels. It may be concluded that the substance is without any mutagenic activity on the bacterial strains used in the test.

ANTIBACTERIAL ACTIVITY
Result show that test item is without antibacterial activity up tn the dose of 5000 µg/plate: counts of CFUs in the control plates and in plates containing the highest dose of compound did not differ significantly.

Mutagenicity

Strain	Dose µg/plate	Revertant colonies + S9-mix		Revertant colonies - S9-mix
		Mean	R*	Mean	R*
TA1535	5000	14	< 2	14	< 2
	1000	11	< 2	16	< 2
	500	14	< 2	13	< 2
	100	12	< 2	13	< 2
	50	13	< 2	15	< 2
	10	13	< 2	11	< 2
	0	12		14
TA1537	5000	7	< 2	6	< 2
	1000	7	< 2	6	< 2
	500	8	< 2	6	< 2
	100	6	< 2	6	< 2
	50	6	< 2	6	< 2
	10	5	< 2	7	< 2
	0	7		6
TA1538	5000	23	< 2	11	< 2
	1000	25	< 2	11	< 2
	500	20	< 2	9	< 2
	100	30	< 2	12	< 2
	50	30	< 2	12	< 2
	10	24	< 2	10	< 2
	0	24		10
TA100	5000	108	< 2	98	< 2
	1000	105	< 2	99	< 2
	500	102	< 2	102	< 2
	100	100	< 2	99	< 2
	50	105	< 2	106	< 2
	10	113	< 2	102	< 2
	0	103		101
TA198	5000	29	< 2	19	< 2
	1000	30	< 2	19	< 2
	500	32	< 2	22	< 2
	100	36	< 2	21	< 2
	50	34	< 2	24	< 2
	10	37	< 2	23	< 2
	0	38		23

* times of increase over the spontaneous reversion rate. Values less than 2 were considered not significant of mutagenic activity.

Antibacterial activity

Strain	Dose µg/plate	Colony counts + S9-mix					Colony counts - S9-mix
		CFU/plate			Mean	% survival	CFU/plate			Mean	% survival
TA1535	0	123	156	170	150	100	130	136	144	137	98
	5000	125	144	186	155		131	133	140	155
TA98	0	210	226	240	225	99	209	240	244	231	98
	5000	207	231	233	223		215	222	241	225

CFU: colony forming units

Control of Salmonella strains

Growth in	TA1535	TA1537	TA1538	TA100	TA98
VB	-	-	-	-	-
VB + his	-	-	-	-	-
VB + bio	-	-	-	-	-
VB + his + bio	+	+	+	+	+
VB + his + bio + amp	-	-	-	+	+
rfa character^a)	20	22	20	21	20
uvrB character^b)	high	high	high	high	high
Spontaneous reversion rate^c)	10, 14, 16	4, 4, 7	8, 16, 17	95, 100, 171	9, 15, 31
Reversion by
2-aminofluorene 10 µg + S9-mix	11, 13, 17	46, 48, 54	> 1000^d)	> 1000^d)	> 1000^d)
methylmethanesulfonate 1000 µg	10, 11, 17	3, 7, 8	19, 12, 15	> 1000^d)	18, 22, 27
ethylmethanesulfonate 5000 µg	> 1000^d)	4, 5, 7	20, 22, 23	> 1000^d)	18, 22, 34
9-aminoacridine 10 µg	12, 13, 14	237, 315, 322	9, 15, 18	122, 138, 160	9, 21, 24

^a) mm of the diameter of inhibition zone caused by 10 µg of crystal violet

^b) high or low sensibility to ultraviolet irradiation

^c) number of revertant colonies per plate

^d) result from three different plates

Control of S9 -mix

Sterility	OK
Activity	Number of revertants/plate
TA1538	28, 11, 32
TA1538 + S9-mix	42, 40, 51
TA1538 + AF (10 µg)	34, 42, 47
TA1538 + AF (10 µg) + S9-mix	> 1000, > 1000, > 1000

AF = 2-aminofluorene

Conclusions:

The substance did not showed any mutagenic activity on the bacterial strains used in the test, with and without metabolic activation.

Executive summary:

Ames strains of Salmonella typhimurium TA1535, TA1537, TA1538, TA100 and TA98 were tested in order to assess the test item mutagenicity activity.

A first experiment has been performed on TA1535 and TA98 to evaluate the antibacterial activity potential at the concentration of 5000 µg/plate; after that, the mutagenicity assay has been performed on all strains at concentrations between 5000 - 10 µg/plate.

Result show that test item is without antibacterial activity up to the dose of 5000 µg/plate: counts of CFUs in the control plates and in plates containing the highest dose of compound did not differ significantly.

None of the tested doses of test item increases the number of revertants over the spontaneous levels.

Conclusion

The substance did not showed any mutagenic activity on the bacterial strains used in the test, with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Non mutagenic

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Study period:

From March 10th to May 06, 1986

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:

Justification for Read Across is detailed in 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:

May 26, 1983

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KIeintierfarm Madoerin AG, CH 4414 Fuellinsdorf/Switzerland.
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: males 31 - 38 g, females 25 - 32 g.
- Housing: n Makrolon type-3 cages with wire mesh top and granulated softwood bedding.
- Diet: peIIeted standand Kliba no. 343 mouse diet, Batch 33/85 (Klingentalmuehle), ab Iibitum.
- Water: tap water, ad libitum.
- Acclimation period: 7 days under laboratory conditions, after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidit: 40 - 70 %
- Photoperiod: 12 hours artificial fluorescent light / 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

4 %, w/v carboxymethylcellulose sodium salt in distilled water

Details on exposure:

- Volume administrated: 20 ml/kg body weight

FORMULATION OF THE TEST ARTICLE
A suspension was prepared immediately before application by adding the test article to distilled water containing CMC (4 % w/v).
Homogeneity of the suspension was maintained during application using a magnelic stirrer.

DOSE SELECTION
The maximum tolerated dose based on acute oral toxicity data. A preliminany acute oral LD50 study (Iimit test; 1 dose, 3 males and 3 females per dose) performed with the same mouse strain as used in this study showed the following result after 15 days observation: 5000 mg/kg body weight, 0 mortality in 6 animals. The 5000 mg/kg hody weight was used in the current study as the maximurn tolerated dose.

Frequency of treatment:

Single application

Dose / conc.:

5 000 mg/kg bw/day

No. of animals per sex per dose:

three groups, each containing 18 males and 18 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Vehicle: dissolved in 0.9 %, w/v saline solution.
- Dose: 50 mg/kg body weight

Tissues and cell types examined:

One thousand polychromatic erythrocytes (PCE) per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (NCE) was used to assess the toxicity of the test article by counting a total of 1000 erythrocytes.

Details of tissue and slide preparation:

SACRIFICE
24, 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marrow, was removed from the femora for examination. The first five animals were used for evaluation. AII mice were sacrificed by cervical dislocation. The femora were removed from each mouse and freed of adherent tissue.

PREPARATION OF SUSPENSION
The proximal end of the femur was cut with scissors; the distal end was left intact. The needle of a plastic syringe containing 0.2 ml calf serum was inserted into the proximar part of the marrow canal.
The bone marrow cells were dispersed in 1.5 ml of calf serum as a homogeneous suspension. The tube containing the bone marrow cells of both femora was centrifuged at 1000 RPM (ca. 180-190 G) for 5 minutes.
The supernatant was removed, leaving a thin layer of serum. The cells of the sediment were suspended by aspiration in a siliconized pasteur pipette. A small drop of the marrow, serum suspension was smeared on the slide, which was allowed to dry overnight. Two slides per animal were prepared. One to three days later, the smears were stained using the panoptic stain method developed by Pappenheim.

METHOD OF ANALYSIS AND EVALUATION
The slides were coded before microscopic analysis. If macroscopic evaluation revealed technical imperfections, the first slide was replaced by the second slide prepared. From each animal, one thousand (1000) polychromatic erythrocytes (PCE) were scored under the microscope (magnification 1000×), for the incidence of micronuclei. Micronuclei were evaluated according to the characteristics which have been described by Schmid. Some characteristics of micronuclei are round features of a sharp contour, sometimes slightly oval or ring-shaped, average size of 1/20 to 1/5 of the erythrocyte size and darkly blue-stained, comparable with the staining quality of nucleic material. Structures of irregular or fibrillar shape, of divergent staining properties of other characteristics not typical for a micronucleus (e.g. atypical focus properties) were not considered to be micronuclei.
Multiple micronuclei in one cell were registered and calculated as one micronucleated cell.
The calculated ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), based on 1000 erythrocytes scored per slide, measured the toxic efficacy of the test article.
Polychromatic erythrocytes (PCE) can be differentiated from normochromatic erythrocytes (NCE) by their basophilic, blue- grey color compared to the acidophilic orange-pink colon of NCE. PCE also tend to be somewhat larger and have more diffuse boundaries.
Additional information chromatic erythrocytes could be obtained for micronuclei.

Evaluation criteria:

The frequencies of micronucleated polychromatic erythrocytes of the treated male and female groups were compared with those of the negative control groups at each sampling time. A regression model assuming a Poisson distribution was applied.
Estimation and testing were performed by maximum likelihood method. If a test article produced no statistically significant positive response at any one of the test points, it was considered to be non-mutagenic in this system.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

in males

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

After a single application of the test article at 5000 mg/kg body weight by gavage no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male of female treated groups when compared with corresponding negative control groups. These results were found at the three examination times , 24, 48 and 72 hours post-application, respectively.
Reduced PCE/NCE ratios were observed in bone marrow preparations of male animals 24, 48 and 72 hours post-application. In female animals, no toxic effect of the test article was observed.

POSITIVE CONTROL
The positive control groups receiving cyclophosphamide exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes 24 and 48 hours post-application and thus validated the test. The dose used for this reference mutagen exhibited a toxic effect as should by the reduced PCE/NCE ratio.

CLINICAL EXAMINATION
No death occurred during the application period. Yellowish-coloured urine and faces as well as diarrhea was observed in all test article-treated animals up to 48 hours post-application. In male animals, slight sedation was observed 4 hours and ruffled fun 48 hours post-application.
The animals were free of clinical findings 56 hours post-application and later.

Conclusions:

The test item is not considered to be mutagenic in the in vivo mouse micronucleus assay performed.

Executive summary:

The study with mice was performed to assess the potential mutagenic activity, induced by test item, through damage to the chromosomes on mitotic apparatus, according to OECD guideline 474.

The experiment was performed with three groups, each containing 18 males and 18 females, for a total of 108 mice. The negative control group received the test article vehicle, i.e. 4 %, w/v carboxymethylcellulose sodium salt in distilled water. The test groups received 5000 mg/kg body weight as the maximum tolerated dose of the test article. The positive control groups received 50 mg/kg body weight cyclophosphamide dissolved in 0.9 %, w/v saline solution. All animals were given a single application of 20 ml/kg body weight by gavage.

24, 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marrow, was removed from the femora for examination. The first five animals were used for evaluation. One thousand polychromatic erythrocytes (PCE) per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes (NCE) was used to assess the toxicity of the test article by counting a total of 1000 erythrocytes.

The data were statistically analyzed by means of a regression model assuming a Poisson distribution. Estimation and testing were performed by maximum likelihood method.

Reduced PCE/NCE ratios were observed in bone marrow preparation of male animals 24, 48 and 72 hours post-application. In female animals, no toxic effects of the test article was observed.

After single application of the test article at 5000 mg/kg body weight by gavage, no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male or female treated groups, when compared with corresponding negative control groups. These results were found at the three examination times, 24, 48 and 72 hours post-application, respectively. The positive control groups, which received cyclophosphamide, exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes 24 and 48 hours post-application and thus validated the test.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced no chromosome mutations by chromosome-breaking activity or by damage to the mitotic apparatus. Therefore, test item is not considered to be mutagenic in the in vivo mouse micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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 vivo chromosomal aberration assay.

The gene mutation assays in bacteria and in mammalian cells were conducted with Basic Orange 064; the in vivo chromosomal aberration test available was conducted on the structural analogue Similar Substance 02; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

IN VITRO BACTERIA GENE MUTATION ASSAY

Two experiments assaying the bacteria gene mutation conducted with Basic Orange 064 are available; one was conducted in the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 100 and TA 98; the other one was conducted using Escherichia coli strain WP2 uvrA.

Result show that test item is without antibacterial activity up to the dose of 5000 µg/plate. None of the tested doses of test item increases the number of revertants over the spontaneous levels in both Salmonella typhimurium and Escherichia coli strains.

In addition, the available information on the structural analogous Similar Substance 01 have been taken into consideration. Similar Substance 01 is the acid form of Basic Orange 064 (details about the approach are reported into the IUCLID section 13). Back mutation test has been performed according to testing method described by Ames (1975) and Tajima et al. (1980). Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 1538, TA 98 and Escherichia coli WP2 uvrA strains were used. No remarkable increase in the mutation colony was recognized. Therefore, the specimen was judged not to posses mutagenicity concerning the test conducted.

IN VITRO MAMMALIAN CELL GENE MUTATION ASSAY

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

The main experiment showed that Basic Orange 064 over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control) with and without metabolic activation. Thus, the test item is considered as being non-mutagenic in this system.

IN VIVO CHROMOSOMAL ABERRATION

The available information on the structural analogue Similar Substance 02 have been taken into consideration; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

The study with mice was performed to assess the potential mutagenic activity, induced by Similar Substance 02, through damage to the chromosomes on to the mitotic apparatus, according to OECD guideline 474. The experiment was performed with three groups, each containing 18 males and 18 females, for a total of 108 mice. The test groups received 5000 mg/kg body weight as the maximum tolerated dose of the test article.

Reduced PCE/NCE ratios were observed in bone marrow preparation of male animals 24, 48 and 72 hours post-application. In female animals, no toxic effects of the test article was observed.

After single application of the test article at 5000 mg/kg body weight by gavage, no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male or female treated groups, when compared with corresponding negative control groups. These results were found at the three examination times, 24, 48 and 72 hours post-application, respectively. The positive control groups, which received cyclophosphamide, exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes 24 and 48 hours post-application and thus validated the test.

DISCUSSION AND CONCLUSION

Based on both in vitro and in vivo available information, a genotoxic potential of Basic Orange 064 may be excluded.

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 in vitro and in vivo information suggest that test substance has no potential to induce genetic toxicity.

 

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