5',5'''-[ethylenebis(p-phenyleneazo)]bis[1',2'-dihydro-6'-hydroxy-4'-methyl-2'-oxo-1,3'-bipyridinium] dilactate

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 bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364
Green M.H.L. and Muriel W.J. Mutation Res. (1976) 38, 3 -32

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

The Escherichia coli was obtained from "National Collection of Industrial Bacteries", Torrey Research station P.O., Aberdeen, Scotland, UK.
The strains cultures were kept in sterile 0.5 ml ampoules (0.45 ml bacterial culture + 0.05 ml dimethylsulfoxide) at -70 °C and in liquid nitrogen.
The test strains were checked at regular intervals for their genetic markers.

Starting experiment the bacteria were grown overnight in a shaking waterbath for 16 h at 37 °C using 2.5 % nutrient broth no. 2 (source: Oxoid). After centrifugation the bacteria were resuspended to a titer of about 5 × 10E8 – 3 × 10E9 cells per milliliter in 0.16 % nutrient broth and 0.5 % NaCl. This titer was controlled photometricallly and determined in an experimental test with histidine-rich KCl solution on minimal agar plate.

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate

Untreated negative controls:

yes

Remarks:

aqua bidest.

True negative controls:

yes

Remarks:

aseptic control with test material and S-9 mix

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
Immediately before the experiment the highest dose solution was prepared by adding the test material to the appropriate solvent. The other doses were dilutions of the high dose with the solvent. The following materials were mixed in a test tube and poured onto minimal agar plates:
100 µl tests solution or control solvent or positive control solution
500 µl S-9 mix (for tests with metabolic activation) or Na2HPO4, 0.15 M (for tests without metabolic activation)
100 µl Bacteria suspension (1 × 10E8 – 2 × 10E9 cells/ml)
2 ml Molten agar consisting of 0.6 % Bacto agar and 0.6 % NaCl supplemented with 10 % 0.5 mM L-histidine and 0.5 biotine solution.
A pourmatic was used to fill the plates automatically with 22 ml of 1.5 % Bacto agar in Vogel Bonner medium E with 2 % glucose.

DURATION
Exposure duration: plates were incubated in the dark for 3 days.

NUMBER OF REPLICATIONS
Each compound concentration, including controls, was tested in triplicate.

NUMBER OF CELLS EVALUATED
1.6 - 3.1 × 10E9 cells/ml

ASEPTRIC CONTROL
For the aseptic control experiments 100 µl of the solution of the test compound or 100 µl of S-9 mix were added to 2 ml molten agar and treated as described above.

DETERMINATION OF CYTOTOXICITY
The toxicity of the test material may be determined by a reduction of the number of spontaneous revertants and by an examination of the background lawn of bacterial growth resulting from traces of tryptophane added to the top agar. Toxicity reduces the sensitivity to testing of mutagenicity in a bacterial test. Therefore, the toxicity estimation is required to validate the collected data.

OTHER EXAMINATIONS
The his+ revertant colonies were counted with a Fisher counter 880 (Fisher, Comp).

TEST CONDITIONS
All experimentation was carried out under sterile conditions.
In order to avoid any light effects on labile test compounds, all experimentation was carried out under yellow light.

LIVER MICROSOMAL FRACTION S-9 MIX
For the study fresh liver preparations from animals, sacrificed on the day of the experiment, were used.
Specific pathogen-free male Wistar rats (180 - 250 g outbred) were obtained from Kleintierfarm Madoerin AG, Fuellinsdorf/BL, Switzerland. After acclimatization the rats received five days before the experiment a single ip. injection of Aroclor 1254 (source: Analabs) dissolved in oleum arachidis (200 mg/ml) at a dosage of 500 mg/kg body weight to induce liver microsomal enzyme activity. The rats were killed on the fifth day p. appl. after a 14 - 16 hour starvation period.
The livers were removed under aseptic conditions and homogenised with 0.15 molar, ice cold KCl (5 g of liver to 15 g of KCl). The homogenates were centrifuged at 9000 g for 10 minutes at 0 to 2 degrees centigrade. The supernatant fraction (S-9 fraction) was collected for the preparation of S-9 mix.
Composition of 1 ml S-9 mix
Na2HPO4100 µmoles
MgCl28 µmoles
KCl 33 µmoles
NaDP+4 µmoles
G-6-P 5 µmoles
S-9 fraction 0.3 ml

Evaluation criteria:

A mutagenic activity was assumed if at least a two fold increase of the number of induced revertants was obtained in comparison with the spontaneous revertants of the corresponding controls.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A slight precipitation of the test material was observed after pouring the content of the test tube onto the surface of the selective agar plate at the concentration above 158 µg/plate. The revertants were easily recognized, therefore the plates could be evaluated (counting by hand).
Up to the highest tested dose, with and without microsomal activation, no relevant increase of the revertant colony numbers was obtained in comparison with the corresponding controls.

TOXICITY OF THE TEST MATERIAL
No toxic effects of the test item were observed.

RESULTS OF CONTROL EXPERIMENTS
The control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate within the normal range of the testing laboratory experience.
The control plates with reference mutagens (positive controls) showed a distinct elevation of the revertant colonies with the tester strain. The positive results of the mutagen 2-aminoanthracene indicate that the metabolizing system was functioning.

The aseptic control showed no contamination for either the test material solution or for the S-9 mix.

Conclusions:

In the experiments performed, no relevant increase of the revertant colony numbers was observed in Echerichia coli strain tested, in the presence and in the absence of S-9 mix.

Executive summary:

The compound was tested for detecting its potential gene mutagenic activity according to the plate incorporation method described by Green and Muriel (1976) and Ames et al (1975), using the Escherichia coli strain WP2 uvrA. The tests was performed with and without metabolic activation. The compound was examined in triplicate for 8 concentrations from 1.58 to 5000 µg/plate.

A slight precipitation of the test material was observed after pouring the content of the test tube onto the surface of the selective agar plate at the concentration above 158 µg/plate. The revertants were easily recognized, therefore the plates could be evaluated.

Up to the highest tested dose, with and without microsomal activation, no relevant increase of the revertant colony numbers was obtained in comparison with the corresponding controls.

No toxic effects of the test material were observed.

Conclusion

In the experiments performed, no relevant increase of the revertant colony numbers was observed in Echerichia coli strain tested, in the presence and in the absence of S-9 mix.

REFERENCE

Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364

Green M.H.L. and Muriel W.J. Mutation Res. (1976) 38, 3 -32

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA1535, TA1537 and TA1538

Details on mammalian cell type (if applicable):

All strains contain a histidine mutation and two additional mutations which increase their sensitivity to mutagens. One mutation causes loss of the excision repair system uvrB, the other causes loss of the lipopolysaccharide barrier that coats the surface of the bacteria (rfa). The two strains TA 98 and TA 100 contain a resistance transfer factor (r-factor) which renders both extremely sensitive in detecting several mutagens. The strains TA 1535 and TA 100 are sensitive in detecting mutagens causing base-pair substitutions, the strains TA 1537, TA 1538 and TA 98 are sensitive in detecting mutagens causing frameshift mutations.

The strain cultures were kept in sterile 0.5 ml ampoules (0.45 ml bacterial culture + 0.05 ml dimethylsulfoxide) at -70 °C and in liquid nitrogen.
The test strains were checked at regular intervals for their genetic markers according to the directions in the literature (Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364).

Starting experiment the bacteria were grown overnight in a shaking waterbath for 16 h at 37 °C using 2.5 % nutrient broth no. 2 (source: Oxoid). After centrifugation the bacteria were resuspended to a titer of about 1 × 10E8 – 2 × 10E9 cells per milliliter in 0.16 % nutrient broth and 0.5 % NaCl. This titer was controlled photometricallly and determined in an experimental test with histidine-rich KCl solution on minimal agar plate.

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate

Untreated negative controls:

yes

Remarks:

aqua bidest.

True negative controls:

yes

Remarks:

aseptic control with test material and S-9 mix

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
Immediately before the experiment the highest dose solution was prepared by adding the test material to the appropriate solvent. The other doses were dilutions of the high dose with the solvent. The following materials were mixed in a test tube and poured onto minimal agar plates:
100 µl tests solution or control solvent or positive control solution
500 µl S-9 mix (for tests with metabolic activation) or Na2HPO4, 0.15 M (for tests without metabolic activation)
100 µl Bacteria suspension (1 × 10E8 – 2 × 10E9 cells/ml)
2 ml Molten agar consisting of 0.6 % Bacto agar and 0.6 % NaCl supplemented with 10 % 0.5 mM L-histidine and 0.5 biotine solution.
A pourmatic was used to fill the plates automatically with 22 ml of 1.5 % Bacto agar in Vogel Bonner medium E with 2 % glucose.

DURATION
Exposure duration: plates were incubated in the dark for 3 days.

NUMBER OF REPLICATIONS
Each compound concentration, including controls, was tested in triplicate.

NUMBER OF CELLS EVALUATED
3.2 × 10E8 – 2.2 × 10E9 cells/ml

ASEPTRIC CONTROL
For the aseptic control experiments 100 µl of the solution of the test compound or 100 µl of S-9 mix were added to 2 ml molten agar and treated as described above.

DETERMINATION OF CYTOTOXICITY
To estimate the toxicity of the test material, prototrophic bacteria (his+ spontaneous revertants from TA 1537) were used. These bacteria were added as an internal standard to plates together with the bacteria strain TA 1537 which gives low numbers of revertant colonies (this mixture is referred to as RTA) and their survival was determined. The ratio of the differences in the numbers of colonies of the RTA and the TA 1537 plates for each substance concentration and solvent control gives the relative survival rate.
In addition, the toxicity of the test material may be determined by a reduction of the number of spontaneous revertants in the tests with the inserted strains, and by an examination of the background lawn of bacterial growth resulting from traces of histidine added to the top agar. Toxicity reduces the sensitivity to testing of mutagenicity in a bacterial test. Therefore, the toxicity estimation is required to validate the collected data.

OTHER EXAMINATIONS
The his+ revertant colonies were counted with a Fisher counter 880 (Fisher, Comp).

TEST CONDITIONS
All experimentation was carried out under sterile conditions.
In order to avoid any light effects on labile test compounds, all experimentation was carried out under yellow light.

LIVER MICROSOMAL FRACTION S-9 MIX
For the study fresh liver preparations from animals, sacrificed on the day of the experiment, were used.
Specific pathogen-free male Wistar rats (180 - 250 g outbred) were obtained from Kleintierfarm Madoerin AG, Fuellinsdorf/BL, Switzerland. After acclimatization the rats received five days before the experiment a single ip. injection of Aroclor 1254 (source: Analabs) dissolved in oleum arachidis (200 mg/ml) at a dosage of 500 mg/kg body weight to induce liver microsomal enzyme activity. The rats were killed on the fifth day p. appl. after a 14 - 16 hour starvation period.
The livers were removed under aseptic conditions and homogenised with 0.15 molar, ice cold KCl (5 g of liver to 15 g of KCl). The homogenates were centrifuged at 9000 g for 10 minutes at 0 to 2 degrees centigrade. The supernatant fraction (S-9 fraction) was collected for the preparation of S-9 mix.
Composition of 1 ml S-9 mix
Na2HPO4 100 µmoles
MgCl2 8 µmoles
KCl 33 µmoles
NaDP+ 4 µmoles
G-6-P 5 µmoles
S-9 fraction 0.3 ml

Evaluation criteria:

A mutagenic activity was assumed if at least a two fold (for TA 100: one and half fold) increase of the number of induced revertants was obtained in comparison with the spontaneous revertants of the corresponding controls.

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A slight precipitation of the test material was observed after pouring the content of the test tube onto the surface of the selective agar plate at the concentration above 158 µg/plate. The revertants were easily recognized, thus the plates could be evaluated (counting by hand).
Up to the highest tested dose, with and without microsomal activation, no relevant increase of the revertant colony numbers was obtained in any Salmonella thyphimurium strain in comparison with the corresponding controls.

TOXICITY OF THE TEST MATERIAL
The toxicity of the compound was tested over a series of 8 concentrations; neither quantitative nor qualitative evidence of a toxic effect of the compound was observed.

RESULTS OF CONTROL EXPERIMENTS
The control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate within the normal range of the testing laboratory experience and similar to those described in literature (Ames et al, 1975).
The control plates with reference mutagens (positive controls) showed a distinct elevation of the revertant colonies with the tester strains. This confirmed the reversion properties of each strain. The positive results of the mutagens 2-aminoanthracene and benzo(a)pyrene indicate that the metabolizing system was functioning.

The aseptic control showed no contamination for either the test material solution or for the S-9 mix.

Conclusions:

In the experiments performed, no relevant increase of the revertant colony numbers was observed in any Salmonella typhimurium strains tested, in the presence and in the absence of S-9 mix

Executive summary:

The compound was tested for detecting its potential gene mutagenic activity according to the plate incorporation method of Ames et al (1975), using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. The tests were performed with and without metabolic activation. The compound was examined in triplicate for 8 concentrations from 1.58 to 5000 µg/plate.

A slight precipitation of the test material was observed after pouring the content of the test tube onto the surface of the selective agar plate at the concentration above 158 µg/plate. The revertants were easily recognized, therefore the plates could be evaluated.

Up to the highest tested dose, with and without microsomal activation, no relevant increase of the revertant colony numbers was obtained in any Salmonella thyphimurium strain in comparison with the corresponding controls.

No toxic effects of the test material were observed.

Conclusion

In the experiments performed, no relevant increase of the revertant colony numbers was observed in any Salmonella typhimurium strains tested, in the presence and in the absence of S-9 mix.

REFERENCE

Ames BN, Mc Cann J, Yamasaki E, Mutation Res. (1975) 31, 347 - 364

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From September 26th to October 12th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Strain: Sub-line (K1) of Chinese hamster ovary cell line CHO; the CHO K1 is a sub-line of CHO cell line.
- Lot. No.: 12G006
- Supplier: ECACC (European Collection of Cell Cultures).
- Storage: the cell stocks are kept in liquid nitrogen.
- Health check: 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; results were fully documented within the raw data file.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction

Test concentrations with justification for top dose:

125, 250, 500, 1000 and 2000 µg/ml

Vehicle / solvent:

- Vehicle used: Ham's F12 medium.
- Justification for choice of solvent/vehicle: solvent was chosen based on the results of the preliminary solubility test and the suitability is confirmed with the available testing laboratory’s historical database.

Untreated negative controls:

yes

Remarks:

Ham's F12 medium

Negative solvent / vehicle controls:

yes

Remarks:

Ham's F12 medium and DMSO

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

FORMULATION
- Stock solution: test item was prepared in a concentration of 10 mg/ml with Ham's F12 medium.
- Correction factor: 1.199

METHOD OF APPLICATION: in medium.
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. The CHO K1 cells 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 %).

PROCEDURES
- Exposure duration: 5-hour treatment in the presence and absence of S9-mix was performed.
- Cells number: 5 ×10^6 cells.
- Replicate: duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix.
- Incubation: for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
- Post-exposure treatment: the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution.
- Plating for survival: in samples where sufficient cells survived, cell number was adjusted to 10^5 cells/ml. Samples from these cultures were diluted to 40 cells/ml.

REPLICATES: 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).

INCUBATION: the dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for growing colonies.

EXPRESSION 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 AGENT: 6-thioguanine (3.4 µg/ml). 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 selection agent.

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.

COUNT: cells were counted using a Bürker chamber.

METABOLIC ACTIVATION
- System: S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- Supplier: Trinova Biochem GmbH.
- S9 mix composition: N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid 0.2 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.

DETERMINATION OF CYTOTOXICITY
- Preparation: the cultures was trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells was seeded into petri dishes at 5×10^6 cells each and incubated with culture medium.
- Treatment: after 24 hours the cells were treated with 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.
- Dishes and incubation: 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.
- Assessment: in order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control.
- Observations: 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.

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:

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.

The test item 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:

no 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.

OTHER OBSERVATIONS
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 homogeneous suspension was obtained up to a concentration of 10 mg/ml. For examined test item concentrations no precipitation in the medium was noted.

SUMMARIZED RESULTS OF THE PRE-TEST ON TOXICITY (5-HOUR TREATMENT WITH AND WITHOUT S9-MIX)

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	202	198	204	201.3	100
Test item	62.5	–	5	203	204	205	204.0	101
	125	–	5	205	201	201	202.3	100
	250	–	5	199	203	204	202.0	100
	500	–	5	204	204	200	202.7	101
	1000		5	201	197	199	199.0	99
	2000	–	5	150	152	148	150.0	75
Solvent control (Ham's F12 medium)	–	+	5	201	203	201	201.7	100
Test item	62.5	+	5	205	200	202	202.3	100
	125	+	5	203	198	199	200.0	99
	250	+	5	200	201	204	201.7	100
	500	+	5	198	202	202	200.7	100
	1000	+	5	199	196	195	196.7	98
	2000	+	5	194	192	196	194.0	96

^a Relative to solvent control

Conclusions:

The test item was not mutagenic in the in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.

Executive summary:

The test item was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was suspended 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: 125, 250, 500, 1000 and 2000 µg/ml.

In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration.

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 and 7, 12-Dimethyl benzanthracene caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

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

Conclusion

It is concluded that the test item was not mutagenic in the in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 28th to December 15th, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Principles of method if other than guideline:

Chromosomal aberration test using Chinese Hamster's lung cells was conduced on test item. Cells were cultured for 24 and 48 hours. Two hours before collecting cells, colcemid was added so that cells were colled at the intermediate phase of division. Cells of the third day of culture were treated with trypsin and S9 mix was added. After 3 hours, cells were further cultured for 21 hours; then, the cells at the intermedfiate phase of division were collected.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese Hamster's lung

Details on mammalian cell type (if applicable):

No micoplasm.
The culturing liquor of which 10 % calf serun (Difco) is added to Eagle MEM (Difco) is used.
The cell in a plastic glass-schale (Falcon) is cultured in a CO2-incubator at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0.056, 0.11, 0.23, 0.45 mg/ml

Vehicle / solvent:

Physiological salt (saline)

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

mitomycin C

Details on test system and experimental conditions:

PREPARATION OF DOSING SOLUTION
After the sample is dissolved in physiological salt solution, it is filtered by millipour filter to reduce bacteria and then used.

PRELIMINARY TEST (Making of cell multiplication obstruction curve)
Cell at logarithmic multiplication phase was trypsin treated and collected. 6 × 10^3 of cells were put into 30 mm schale with culturing liquor and cultured. After 3 days the cells were treated to a specimen and collected on the fifth day and the number of cells was counted by hemacytometer. 3 schales each were used in the experiment and their average was taken. The concentration which obstructs multiplication at 50 % was decided to be maximum test concentration.

CHROMOSOMAL ABERRATION TEST
Cells at logarithmic multiplication phase are 0.25 % trypsin treated and collected. 4 × 10^3 of cells are sprinkled in 60 mm schale and cultured for 72 hours. After culturing, the test is done with the concentration determined by the preliminary test as the maximum. Regarding positive control, each is tested at 50 % obstruction concentration.

- Without metabolic activation assay
Specimen is added to 5 ml of culture liquor and cells are cultured for 24 and 48 hours. Two hours before collecting cells, colcemid is added so that 0.2 µg/ml is achieved and cells at the intermediate phase of division are collected.

- With metabolic activation assay
Cells at the third day of culture is trypsin treated and adjusted to 10^7 Cells/ml. 0.2 ml cells, specimen 0.2 ml and S-9 mix 1 ml are added and lightly shaken in a water bath at the constant temperature of 37 °C. After three hours, it is centrifuqally separated and serum is removed. Then the cells are divided into 2 plates of schale (6 cm) and is further cultured for 21 hours and then the cells at the intermediate phase of division are collected.

NUMBER OF REPLICATIONS
Each test is carried out by using 2 plates of schale and done twice independently.

FIXED DYEING
Culture liquor is transferred to centrifugal tube and the cells are collected by centrifugation.
Cells in schale are Trypsin treated and collected and put together with the aforementioned cells. The collected cells are left for 10 minutes in 0.075 M KCl solution at 37 °C. After being fixed by Acetic Acid-Methanol (1:3), the cells are dropped on 2 sheets of acid treated slide glasses and dyed by 1.3 % Giemsa solution.

OBSERVATION
Intermediate phase of division of 100 Cells in each preparation is observed. Aberration of polyploidy, gaps, breaks, exchange, ring formation and fragmentation as well as dicentric in chromatids and others are calculated.

METABOLIC ACTIVATION
Rat S-9 (Kikkoman) is purchased and used.
Preparation of S-9 mix: the composition of S-9 mix is 0.1 ml S-9 and 0.4 ml supplementary gene solution.
0.1 ml of 10^7 cell suspension liquor, 0.1 ml specimen and 0.5 ml S-9 mix are added and metabolic activated.
S-9 mix composition
MgCl2 (50mM) 1 ml
KCl (330 Mm) 1 ml
Hepes buffer solution (20 mM) 2 ml
Glucose-6-Phosphoric Acid (50 mM) 1 ml
NADH (40 mM) 1 ml
Distilled Water 1 ml
S-9 3 ml

Evaluation criteria:

Gaps are not included in chromosomal aberration. As compared with controlled (normally 3 %), a number of chromosome aberration is considered negative if it is within 3 - 5 %; it is pseudo-positive if between 5 - 10 %; and it is positive if it is between 10 - 20 % and furthermore the dosage of effectiveness is recognized. If between 20 - 50 %, it is considered high positive and over 50 % is strongly positive.

Species / strain:

other: Chinese Hamster's lung

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No occurrence of chromosomal aberration when test item was applied both without and with metabolic activation.

Conclusions:

No occurrence of chromosomal aberration was recorded when test item was applied both without and with metabolic activation.

Executive summary:

Chromosomal aberration test using Chinese Hamster's lung cells was conducted on test item. The test was conducted with the concentrations determined by the preliminary test, as the maximum. Cells were cultured for 24 and 48 hours. Two hours before collecting cells, colcemid was added so that cells were colled at the intermediate phase of division. Cells of the third day of culture were treated with trypsin and S9 mix was added. After 3 hours, cells were placed in 2 plates and further cultured for 21 hours; then, the cells at the intermediate phase of division were collected. Aberration of polyploidy, gaps, breaks, exchange, ring formation and fragmentation as well as dicentric in chromatids and others are calculated.

No occurrence of chromosomal aberration was recorded when test item was applied both without and with metabolic activation.

Conclusion

No occurrence of chromosomal aberration was recorded when test item was applied both without and with metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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

IN VITRO BACTERIA GENE MUTATION ASSAY

Basic Yellow 094 was tested for detecting its potential gene mutagenic activity, according to the plate incorporation method of Ames et al (1975) and using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. A slight precipitation of the test material was observed after pouring the content of the test tube onto the surface of the selective agar plate at the concentration above 158 µg/plate. The revertants were easily recognized, therefore the plates could be evaluated. Up to the highest tested dose, with and without microsomal activation, no relevant increase of the revertant colony numbers was obtained in any Salmonella thyphimurium strain in comparison with the corresponding controls. No toxic effects of the test material were observed.

In addition, Basic Yellow 094 was tested using the Escherichia coli strain WP2 uvrA to detect its potential gene mutagenic activity. The test was conducted according to the plate incorporation method described by Green and Muriel (1976) and Ames et al (1975). A slight precipitation of the test material was observed after pouring the content of the test tube onto the surface of the selective agar plate at the concentration above 158 µg/plate. The revertants were easily recognized, therefore the plates could be evaluated. Up to the highest tested dose, with and without microsomal activation, no relevant increase of the revertant colony numbers was obtained in comparison with the corresponding controls. No toxic effects of the test material were observed.

IN VITRO MAMMALIAN CELL GENE MUTATION ASSAY

Basic Yellow 094 was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The objective of the study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells. The main experiment concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study, without and with metabolic activation system. 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 controls was in the range of historical laboratory control data; thus, the study was considered valid. The substance tested up to the maximum recommended concentration, with and without metabolic activation system, over a 5 hour treatment period, did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).

IN VITRO CHROMOSOMAL ABERRATION

Chromosomal aberration test using Chinese Hamster's lung cells was conducted on test item. The test was conducted with the concentrations determined by the preliminary test, as the maximum. Cells were cultured for 24 and 48 hours. Two hours before collecting cells, colcemid was added so that cells were colled at the intermediate phase of division. Cells of the third day of culture were treated with trypsin and S9 mix was added. After 3 hours, cells were placed in 2 plates and further cultured for 21 hours; then, the cells at the intermediate phase of division were collected. Aberration of polyploidy, gaps, breaks, exchange, ring formation and fragmentation as well as dicentric in chromatids and others are calculated.

No occurrance of chromosomal aberration was recorded when test item was applied both without and with metabolic activation.

Justification for classification or non-classification

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

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

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

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

 

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