Ferrate(4-), hexakis(cyano-C)-, methylated 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]benzenamine copper(2+) salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2020 to 12 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The study was performed to investigate the potential of test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation method (Trial I) and the pre-incubation method (Trial II) both in presence and in absence of metabolic activation system, using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ferrate(4-), hexakis(cyano-C)-, methylated 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]benzenamine copper(2+) salts

EC Number:

235-468-7

EC Name:

Ferrate(4-), hexakis(cyano-C)-, methylated 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]benzenamine copper(2+) salts

Cas Number:

12237-62-6

Molecular formula:

[C24N3H28] Cu2Fe(CN)6

IUPAC Name:

ferrate(4-), hexakis(cyano-C)-, methylated 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]benzenamine copper(2+) salts

Test material form:

solid

Remarks:

Violet Powder

Details on test material:

Name of test material: Ferrate(4-), hexakis(cyano-C)-, methylated 4-[(4-aminophenyl) (4-imino-2,5-cyclohexadien-1-ylidene)methyl]benzenamine copper (2+) salts
- CAS No. 12237-62-6
- EC No. 235-468-7
- Molecular formula: [C24N3H28] Cu2Fe(CN)6
- Molecular weight: 697.54 g/mol
- Subsatnce type: Organic
- Physical state: Solid

Method

Target gene:

Histidine Operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Nnot applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Aroclor 1254-induced S9 metabolic activation system.
- source of S9 : Aroclor 1254-induced S9 was procured from the Defence Research and Development Organization
- method of preparation of S9 mix : An appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final concentration of approximately 10 % v/v in the S9 mix. Cofactor solution contains the following quantity of chemicals in 500 mL of Distilled Water.
D-glucose-6-phosphate 0.8 g
β-NADP 1.75 g
MgCl2 1.0 g
KCl 1.35 g
Na2HPO4.H2O 6.4 g
NaH2PO4.H2O 1.4 g
During the experiment, the S9 mix was prepared freshly and used.

- concentration or volume of S9 mix and S9 in the final culture medium : 10 % v/v in the S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data available.

Test concentrations with justification for top dose:

Based on the results of pre-experiment, the following doses were selected for the main study Trial I and Trial II:
0.0 (NC), 0.0(VC), 0.004, 0.012, 0.038, 0.119 and 0.376 mg/plate, both in the absence (-S9) and presence of metabolic activation (+S9).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was found to be soluble at 50 mg/mL to give the final treatment concentration of 5 mg/plate. Hence, DMSO was chosen as a solvent for the study.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Negative Control: Distilled Water Solvent control: DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine [Without metabolic activation (For Strains TA 1537, TA 98)] 2-Aminoanthracene [With metabolic activation (For Strains TA 1535, TA 1537, TA 98, TA 100 and TA 102)]

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : The test chemical was tested in two independent experiments (Trial I and Trial II). Trial I was performed according to plate incorporation method and Trial II was performed according to the pre-incubation method.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Not applicable
- Test substance added in agar (plate incorporation Trial I); preincubation (Trial II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: For 60 minutes at 37 °C
- Exposure duration/duration of treatment: For 48 hours at 37 °C.
- Harvest time after the end of treatment (sampling/recovery times): No data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Bacerial background growth inhibition
- Any supplementary information relevant to cytotoxicity: No data

METHODS FOR MEASUREMENTS OF GENOTOXICIY : For each strain and dose level, including the controls, three plates (triplicate) were used. The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, negative control, vehicle control, and reference mutagen solution (positive control),
500 µL S9 mix (for the test with metabolic activation) or S9 mix substitution buffer (for the test without metabolic activation),
100 µL Bacterial suspension,
2000 µL Overlay agar
In the pre-incubation assay, 100 µL test solution, 500 µL S9 mix and S9 mix substitution buffer and 100 µL bacterial suspensions were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation, 2.0 mL overlay agar (47 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification, the plates were incubated in an inverted position for 48 hours at 37 °C

- OTHER: No data

Rationale for test conditions:

No data available

Evaluation criteria:

A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control was observed.
A dose-dependent increase was considered biologically relevant if the threshold of biological significance was exceeded at more than one concentration.
An increase exceeding the threshold of biological significance at only one concentration was judged as biologically relevant if it was reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies above the threshold of biological significance was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and vehicle control, the increase was not considered biologically relevant.

Statistics:

Microsoft Office Excel-based calculation was used for descriptive statistical analysis.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 0.376 mg/plate (T6): reduction in colony count and moderate inhibition of back ground lawn was observed both in the absence (-S9) and in presence (+S9) of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: Insoluble in water
- Precipitation and time of the determination: Different amounts of formulated test item preparation (50 mg/mL) were added to overlay agar (top agar) in test tubes to give various test item concentration of (maximum 5 mg/plate) and plated on minimal glucose agar (MGA) plates. Precipitation was noticed at 5 mg/plate concentration which was assumed to interfere with the scoring. At treatment concentration, 3.750 mg/plate slight precipitation was observed, which was assumed to be non-interfering with the scoring.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): The pre-experiment was performed with TA 100 and TA 98 strain of Salmonella typhimurium and with eight concentrations spaced by (√10) half-log intervals in triplicates. Based on the solubility and precipitation test, 3.750 mg/plate was selected as the highest dose in the pre-experiment. The following doses were selected for the pre-experiment: 0.0 (NC), 0.0(VC), 0.001, 0.004, 0.012, 0.038, 0.119, 0.376, 1.187 and 3.750 mg/plate. In TA 98 and TA 100 tester strains, the complete inhibition of background lawn and the absence of revertant colonies were observed at 3.750 and 1.187 mg/plate (T8-T7) both in the presence and absence of metabolic activation. Whereas, at 0.376 mg/plate (T6) the, reduction in colony count and moderate inhibition of back ground lawn was observed both in the absence (-S9) and presence (+S9) of metabolic activation. No reduction in colony count and diminution of the background lawn were observed at 0.119 – 0.001 mg/plate (T5-T1) neither in the absence (-S9) nor the presence (+S9) of metabolic activation. Based on the results of the pre-experiment, 0.376 mg/plate was selected as the highest dose for the main study trials both in the absence and presence of metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The spontaneous reversion rates in the negative, vehicle, and positive controls were within the range of in-house historical data range. The positive controls showed distinct increases in the number of revertant colonies in all the tester strains both in the presence and absence of metabolic activation, thus confirming the validity of the assay.

Ames test:
- Signs of toxicity : Increase in the revertant colony count
- Individual plate counts : Please refer the the table attached in remark section.
- Mean number of revertant colonies per plate and standard deviation : Please refer the table attached in any other information on result section.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer the table attached in remark section.
- Negative (solvent/vehicle) historical control data: Please refer the table attached in remark section.

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD TRIAL I

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.00	1.00	12.00	3.00	18.33	4.93	112.67	12.06	241.33	8.33
VC (0.00)	5.67	0.58	16.33	1.53	22.67	4.16	123.33	5.03	265.33	8.33
T1 (0.004)	5.00	1.00	13.67	2.52	19.67	4.51	120.67	7.02	249.33	12.22
T2 (0.012)	5.33	0.58	14.67	1.53	18.67	3.06	116.00	9.85	254.67	14.05
T3 (0.038)	4.33	0.58	15.00	2.00	19.00	3.00	117.33	7.02	244.00	12.00
T4 (0.119)	4.67	0.58	14.00	2.00	21.00	3.61	118.00	5.29	252.00	12.00
T5 (0.376)	0.00	0.00	0.00	0.00	7.33	2.89	28.00	8.19	64.67	10.07
PC	182.67	32.08	416.00	71.11	1234.67	54.45	1221.33	120.36	1472.00	178.12

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	3.67	0.58	11.33	1.53	16.00	2.00	110.00	11.14	236.00	12.00
VC (0.00)	5.33	1.53	14.00	2.00	20.33	3.51	119.33	7.57	260.00	16.00
T1 (0.004)	4.33	0.58	13.33	1.53	18.33	4.16	118.00	4.00	241.33	20.53
T2 (0.012)	4.67	1.15	12.67	2.52	19.00	4.58	117.33	8.08	237.33	10.07
T3 (0.038)	4.00	1.00	11.67	2.08	17.67	3.79	114.00	8.00	248.00	12.00
T4 (0.119)	5.00	1.00	13.00	2.00	18.67	4.04	115.33	5.03	240.00	8.00
T5 (0.376)	0.00	0.00	0.00	0.00	4.00	1.00	30.33	4.51	62.33	4.93
PC	190.67	32.08	1188.00	127.00	970.67	80.53	1109.33	117.12	1672.00	186.03

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  

Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD TRIAL II

Dose (mg/plate)	Presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.67	0.58	12.33	1.53	18.00	3.00	114.00	8.00	240.00	10.58
VC (0.00)	6.00	1.00	16.00	1.00	24.00	1.73	122.67	11.37	262.67	8.33
T1 (0.004)	5.00	0.00	13.33	2.08	21.00	2.65	116.67	7.02	249.33	8.33
T2 (0.012)	4.67	0.58	14.00	2.00	19.67	3.21	117.33	10.07	246.67	12.22
T3 (0.038)	5.67	0.58	15.00	1.00	18.33	1.53	114.67	8.33	244.00	10.58
T4 (0.119)	5.33	0.58	14.33	2.08	20.00	1.00	115.33	9.45	246.00	15.62
T5 (0.376)	0.00	0.00	0.00	0.00	2.67	1.53	27.00	5.00	68.00	4.00
PC	172.00	24.00	432.00	140.63	1304.00	150.04	1300.00	170.27	1228.00	159.80

Dose (mg/plate)	Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.00	0.00	11.33	2.08	17.67	2.08	110.67	7.02	236.00	10.58
VC (0.00)	5.67	0.58	14.00	1.00	22.00	2.00	121.33	9.02	252.00	12.00
T1 (0.004)	4.67	0.58	13.00	1.00	19.33	2.08	115.33	10.07	248.00	12.00
T2 (0.012)	5.00	1.00	13.33	2.08	20.67	2.52	114.00	7.21	244.00	10.58
T3 (0.038)	5.33	0.58	12.33	2.08	18.67	1.53	113.33	9.87	245.33	6.11
T4 (0.119)	4.33	0.58	12.67	2.08	19.00	2.00	114.67	8.33	246.67	10.07
T5 (0.376)	0.00	0.00	0.00	0.00	2.67	1.15	22.33	3.21	57.33	8.74
PC	182.67	22.74	1296.00	153.21	736.00	126.05	1248.00	141.48	1384.00	136.47

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537 [50μg/plate] TA 98 [10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

 

 

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutations in the histidine operon by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system as per the study performed by Ames assay (OECD guideline no. 471).And hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Executive summary:

Ames test as per the OECD guideline No. 471 (Adopted: July 21, 1997, Corrected: June 26, 2020) was performed to investigate the potential of test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation method (Trial I) and the pre-incubation method (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments, both with and without liver microsomal activation. Each concentration, including the negative, vehicle, and positive controls, were tested in triplicate. The pre-experiment was performed with TA 100 and TA 98 strain of Salmonella typhimurium and with eight concentrations spaced by (√10) half-log intervals in triplicates. Based on the solubility and precipitation test, 3.750 mg/plate was selected as the highest dose in the pre-experiment. The concentration 0.0 (NC), 0.0(VC), 0.001, 0.004, 0.012, 0.038, 0.119, 0.376, 1.187 and 3.750 mg/plate doses were selected for the pre-experiment. In TA 98 and TA 100 tester strains, the complete inhibition of background lawn and the absence of revertant colonies were observed at 3.750 and 1.187 mg/plate (T8-T7) both in the presence and absence of metabolic activation. Whereas, at 0.376 mg/plate (T6) the reduction in colony count and moderate inhibition of background lawn was observed both in the absence (-S9) and presence (+S9) of metabolic activation. No reduction in colony count and diminution of the background lawn were observed at 0.119 – 0.001 mg/plate (T5-T1) neither in the absence (-S9) nor the presence (+S9) of metabolic activation. Based on the results of the pre-experiment, 0.376 mg/plate was selected as the highest dose for the main study trials both in the absence and presence of metabolic activation.

Trial-I was performed with five concentrations of the test item along with the negative, vehicle, and concurrent positive controls with the remaining three strains, i.e., TA 1537, TA1535, and TA 102 by the plate incorporation method. For TA 98 and TA 100 tester strains, the revertant colony counts were directly incorporated in the Trial-I from the pre-experiment up to the five concentrations [T2 (0.004 mg/plate) to T6 (0.376 mg/plate)]. The 0.0 (NC), 0.0 (VC), 0.004, 0.012, 0.038, 0.119 and 0.376 mg/plate concentrations of test item were prepared using (√10) half-log intervals, both in the absence (-S9) and presence of metabolic activation (+S9). The plates were treated and incubated at 37°C for 48 hours (approximately). No biologically relevant increase in the revertant count in any of the five strains was observed at any concentrations tested.

Trial-II was performed independently with all the five tester strains along with the negative, vehicle, and positive controls by the pre-incubation method for the confirmation of the Trial-I results. The 0.0 (NC), 0.0 (VC), 0.004, 0.012, 0.038, 0.119 and 0.376 mg/plate concentrations of test item were prepared using (√10) half-log intervals,both in the absence (-S9) and the presence of metabolic activation (+S9) for Experiment -I. The concentration of positive controls used was the same as used in the plate incorporation assay. The test item, negative, vehicle and positive controls were pre-incubated along with 500 µL of metabolic activation mix (+S9) and Buffer (-S9) and 100µL of bacterial culture for 60 minutes at 37 °C in an incubator. After pre-incubation, 2 mL of top agar was mixed with the pre-incubation mixture and poured on minimal glucose agar plates. The treated plates were incubated for 48 hours (approximately) in an incubator. No biologically relevant increase in the revertant count was observed in any of the five tester strains pre-incubated with the test item.The positive controls showed distinct increases in the number of revertant colonies in all the tester strains both in the presence and absence of metabolic activation, thus confirming the validity of the assay.The positive controls showed distinct increases in the number of revertant colonies in all the tester strains both in the presence and absence of metabolic activation, thus confirming the validity of the assay.

From the above data it can be concluded that, the test chemical did not induce gene mutations in the histidine operon by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system as per the study performed by Ames assay (OECD guideline no. 471) and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.