Ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate 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:

January 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: * OECD No. 471 (July 21, 1997) * EEC Directive 92/69, B14 and B13 (December 1992)

Principles of method if other than guideline:

Standard plate test and Preincubation test was performed to determine the mutagenic nature of the test chemical

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)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts

EC Number:

235-469-2

EC Name:

Ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts

Cas Number:

12237-63-7

Molecular formula:

C34H31N8O3Cu3Fe

IUPAC Name:

ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of substance:ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts
- Appearance : red powder
- Common name: Pigment red 169
- Substance type: UVCBs-organometallic

Method

Target gene:

Histidine for Salmonella strains and Tryptophan for E. coli strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

other: histidine auxotrophs (his-)

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

other: tryptophan auxotrophy (trp-)

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

0, 20, 100, 500, 2,500 or 5,000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was selected as the vehicle, which had been
demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Additional plates are treated with soft agar, S-9 mix, buffer, vehiele or the test substance but without the addition of tester strains

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

with S9

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

strains: TA 1535, TA 100, TA 1537, TA 98: 2.5 µg/plate, dissolved in DMSO; Strain: Escherichia coli WP2 uvrA: 60 µg/plate, dissolved in DMSO

Controls 2

Untreated negative controls:

yes

Remarks:

Additional plates are treated with soft agar, S-9 mix, buffer, vehicle or the test substance but without the addition of tester strains

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without S9

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosuguanidine (MNNG)

Remarks:

Strains: TA 1535, TA 100: 5 µg/plate, dissolved in DMSO

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

Strain: TA 98: 10 µg/plate, dissolved in DMSO

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain: TA 1537: 100 µg/plate, dissolved in DMSO

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Strain: E. coli WP2 uvrA: 10 µg/plate, dissolved in DMSO

Details on test system and experimental conditions:

Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix)

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: For PIT: 20 mins
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): 48 - 72 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Rationale for test conditions:

No data

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:

A dose-related and reproducible increase in the nuinber of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adcling a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:

The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Precipitation of the test substance was found from about 500 µg/plate onward.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Standard plate assay

Table: Mutagenic potential

Strain TA1535 Without S9

 

Dose	Rev	M	SD	FAC
DMSO	16	20	3	1.0
	21
	22
20 µg	20	20	1	1.0
	19
	20
100 µg	21	20	1	1.0
	19
	19
500 µg	18P	19	1	1.0
	20P
	19P
2500 µg	5P	5	1	0.3
	4P
	6P
5000 µg	5P	3	2	0.2
	3P
	1P
MNNG 5.0µg	1004	1002	24	50.9
	977
	1024
2-AA 2.5 µg

Strain TA1535 With S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	18	19	2	1.0	29
	18				22
	21				27
20 µg	19	18	1	1.0
	18
	18
100 µg	15	14	1	0.7
	13
	14
500 µg	17P	17	2	0.9
	19P
	19P
2500 µg	7P	6	2	0.3	15
	8P				14
	4P				20
5000 µg	0P				0
	0P				0
	0P				0
2-AA	176	171	14	9.0
	155
	181

 

 

Strain TA100 Without S9

 

Dose	Rev	M	SD	FAC
DMSO	124	115	8	1.0
	108
	113
20 µg	104	101	8	0.9
	107
	94
100 µg	98	102	7	0.9
	110
	99
500 µg	55P	60	5	0.5
	61P
	65P
2500 µg	11P	8	3	0.1
	5P
	8P
5000 µg	0P
	0P
	0P
MNNG 5.0µg	1312	1319	49	11.5
	1273
	1371
2-AA 2.5 µg

 

Strain TA100 With S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	112	111	9	1.0	22
	102				21
	120				29
20 µg	110	110	2	1.0
	108
	111
100 µg	104	114	8	1.0
	118
	119
500 µg	57P	52	17	0.5
	66P
	33P
2500 µg	5P	5	3	0.0	1
	8P				2
	2P				5
5000 µg	0P				0
	0P				0
	0P				0
2-AA 2.5 µg	1135	1274	180	11.4
	1477
	1210

 

Strain TA1537 Without S9

 

Dose	Rev	M	SD	FAC
DMSO	9	10	1	1.0
	10
	10
20	9	9	1	1.0
	9
	10
100	9	10	1	1.0
	10
	11
500	5P	5	2	0.6
	7P
	4P
2500	1P	1	0	0.1
	1P
	1P
5000	0P
	0P
	0P
AAC 100 µg	621	649	27	67.2
	674
	653
2-AA 2.5 µg

 

Strain TA1537 With S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	11	11	1	1.0	38
	11				29
	10				28
20 µg	9	9	2	0.9
	8
	11
100 µg	7	8	1	0.8
	9
	9
500 µg	8P	8	0	0.8
	8P
	8P
2500 µg	1P	1	1	0.1	2
	1P				5
	2P				1
5000 µg	0P				0
	0P				0
	0P				0
AAC 100 µg
2-AA 2.5 µg	173	185	13	17.5
	198
	185

 

Strain TA 98 Without S9

 

Dose	Rev	M	SD	FAC
DMSO	20	27	7	1.0
	29
	33
20 µg	24	21	3	0.8
	19
	21
100 µg	21	23	2	0.9
	24
	25
500 µg	21P	20	1	0.7
	19P
	19P
2500 µg	2P	3	1	0.1
	3P
	4P
5000 µg	0P
	0P
	0P
NOPD 10 µg	937	945	25	34.6
	925
	973
2-AA 2.5 µg

 

Strain TA 98 With S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	42	38	3	1.0	17
	36				24
	36				24
20 µg	35	33	2	0.9
	31
	32
100 µg	34	33	2	0.9
	31
	34
500 µg	20P	22	2	0.6
	24P
	23P
2500 µg	11P	8	3	0.2	2
	8P				2
	6P				2
5000 µg	0P				0
	0P				0
	0P				0
NOPD 10 µg
2-AA 2.5 µg	936	943	13	24.8
	925
	958

 

 

StrainE.coli WP2uvrA Without S9

 

Dose	Rev	M	SD	FAC
DMSO	36	36	1	1.0
	37
	35
20 µg	31	32	4	0.9
	37
	29
100 µg	34	32	3	0.9
	33
	29
500 µg	29P	32	3	0.9
	31P
	35P
2500 µg	25P	28	3	0.8
	30P
	30P
5000 µg	15P	18	3	0.5
	17P
	21P
ENNG 10 µg	999	1009	18	28.0
	998
	1029
2-AA 60 µg

 

Strain E.coli WP2 uvrAWith S9

Dose	Rev	M	SD	FAC	TITRE
DMSO	39	40	2	1.0	40
	42				45
	39				39
20 µg	35	32	3	0.8
	31
	29
100 µg	37	35	3	0.9
	37
	31
500 µg	31P	31	1	0.8
	31P
	30P
2500 µg	20P	24	4	0.6	8
	26P				6
	27P				7
5000 µg	12P	12	1	0.3	0
	12P				0
	13P				0
ENNG 10 µg
2-AA 60 µg	326	314	40	7.9
	347
	269

Applicant's summary and conclusion

Conclusions:

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay with and without metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The study was performed using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. The test chemical was dissolved in DMSO and used at dose level of 0, 20, 100, 500, 2,500 or 5,000 µg/plate. Concurrent solvent and positive control chemicals were also included in the study. According to the results of the present study, the test substance pigment red 169 is not mutagenic in the Salmonella typhimurium /Escherichia coli reverse mutation assay with and without metabolic activation.