Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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 July 1993 to 04 Oct 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

EC Number:

279-365-5

EC Name:

Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Cas Number:

80010-51-1

Molecular formula:

C29H28Cl2N4O7S.Na

IUPAC Name:

sodium 1-amino-4-[(3,5-bis{[(chloroacetyl)amino]methyl}-2,4,6-trimethylphenyl)amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Test material: Polar blau RLS roh trocken (FAT 21036/C)
Batch No.: 410830.32
Purity: about 90 %
Expiration date: June 1998

Specific details on test material used for the study:

Name: FAT 21036/C
Purity: Approx 90 %

Method

Target gene:

Histidine for Salmonella

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):

CELLS USED
- Type and source of cells: Histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 102, TA 1535 and TA 1537) were obtained from Prof. B.
Ames, Berkeley, CA., U.S.A
Strain TA 100 was obtained from Dr. M.Schiipbach, Hofmann-La Roche Ltd., Basle, Switzerland.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction): Rat-liver microsomal fraction S9 was prepared in advance from male RAI rats (Tif: RAIf[SPF]), reared at the Animal Farm of CIBA GEIGY, Sisseln, Switzerland. The animals (150-250 g) were treated with Aroclor 1254 (500 mg/kg, i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl and the 9000x g supernatant (S9) was stored at approximately -80 °C for no longer than one year. The protein contents of the S9 fractions were 33.6 and 30.2 mg/ml.

Test concentrations with justification for top dose:

Range in the cytotoxicity test; 20.6, 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate
Range in the mutagenicity test: 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate

Vehicle / solvent:

Bidistilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For TA 100 & TA 1535 without microsomal activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

For TA 102 without microsomal activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For TA 98 without microsomal activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For TA 1537 without microsomal activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2- aminoanthracene

Remarks:

For TA 100, TA 102, TA 98& TA 1537 with microsomal activation

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

For TA 1535 with microsomal activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY : Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Colonies were counted electronically with an Artek counter

Evaluation criteria:

Assay acceptance criteria:
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the study director.

Criteria for a positive response:
The test substance is considered to be mutagenic in this test system if the following conditions are met:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. Generally a concentration-related effect should be demonstrable.

Statistics:

In deviation to the OECD guideline. a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

RANGE-FINDING/SCREENING STUDIES

Six concentrations of Polar blau RLS roh trocken (FAT 21036/C) ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. The numbers of revertant colonies was not reduced. Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

 

Mutagenicity test, original experiment

In the original experiment carried out without metabolic activation, treatment of strain TA 102 with Polar blau RLS roh trocken (FAT 2103 6/C) led to a marginal increase in the number of backmutants at the concentrations of 1666.7 and 5000 µg/plate. No effects were observed with the other strains. In the experiment with activation performed on strain TA 102, a marginal increase in the number of revertants occurred at the concentration of 555.6 µg/plate only. No effects were observed with the other strains.

 

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without microsomal activation, after treatment with Polar blau RLS roh trocken (FAT 21036/C), no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. The effect observed in the original experiment on strain TA 102 could not be reproduced. In the experiment with activation performed on strain TA 102, a marginal increase in the number of back-mutants occurred at the concentration of 1666.7 µg/plate only. Again, no effects were observed with the other strains. In the mutagenicity tests without and with metabolic activation, normal back-ground growth was observed. The numbers of revertant colonies was not reduced. The test material exerted no toxic effect on the growth of the bacteria. The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

Original experiments without metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	126.3
	61.7284 µg/plate	141.3
	185.1852 µg/plate	155.0
	555.5556 µg/plate	143.0
	1666.6667 µg/plate	144.3
	5000.0000 µg/plate	108.7
	Positive control	1320.3

 

Strain	Treatment	Mean counts
TA 1535	Negative control	13.7
	61.7284 µg/plate	14.7
	185.1852 µg/plate	11.7
	555.5556 µg/plate	11.7
	1666.6667 µg/plate	14.7
	5000.0000 µg/plate	11.33
	Positive control	1100.7

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	15.0
	61.7284 µg/plate	16.0
	185.1852 µg/plate	15.3
	555.5556 µg/plate	21.0
	1666.6667 µg/plate	14.7
	5000.0000 µg/plate	13.0
	Positive control	1582.7

 

Strain	Treatment	Mean counts
TA 1537	Negative control	9.7
	61.7284 µg/plate	8.0
	185.1852 µg/plate	9.0
	555.5556 µg/plate	4.3
	1666.6667 µg/plate	5.0
	5000.0000 µg/plate	5.0
	Positive control	2091.7

 

Strain	Treatment	Mean counts
TA 102	Negative control	237.0
	61.7284 µg/plate	240.0
	185.1852 µg/plate	273.3
	555.5556 µg/plate	327.3
	1666.6667 µg/plate	394.3
	5000.0000 µg/plate	378.0
	Positive control	1130.3

 

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

Original experiments with metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	135.3
	61.7284 µg/plate	130.3
	185.1852 µg/plate	142.7
	555.5556 µg/plate	173.0
	1666.6667 µg/plate	159.3
	5000.0000 µg/plate	144.3
	Positive control	1389.7

 

Strain	Treatment	Mean counts
TA 1535	Negative control	17.0
	61.7284 µg/plate	17.7
	185.1852 µg/plate	14.7
	555.5556 µg/plate	15.3
	1666.6667 µg/plate	14.3
	5000.0000 µg/plate	10.7
	Positive control	339.7

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	34.0
	61.7284 µg/plate	33.7
	185.1852 µg/plate	29.3
	555.5556 µg/plate	31.3
	1666.6667 µg/plate	23.3
	5000.0000 µg/plate	23.7
	Positive control	2207.3

 

Strain	Treatment	Mean counts
TA 1537	Negative control	7.3
	61.7284 µg/plate	9.0
	185.1852 µg/plate	12.0
	555.5556 µg/plate	11.3
	1666.6667 µg/plate	7.3
	5000.0000 µg/plate	4.0
	Positive control	205.7

 

Strain	Treatment	Mean counts
TA 102	Negative control	244.7
	61.7284 µg/plate	348.3
	185.1852 µg/plate	295.3
	555.5556 µg/plate	389.3
	1666.6667 µg/plate	349.0
	5000.0000 µg/plate	354.0
	Positive control	1431.7

 

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

Confirmatory experiments without metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	173.3
	61.7284 µg/plate	195.7
	185.1852 µg/plate	200.3
	555.5556 µg/plate	174.3
	1666.6667 µg/plate	194.3
	5000.0000 µg/plate	183.3
	Positive control	1345.0

 

Strain	Treatment	Mean counts
TA 1535	Negative control	19.0
	61.7284 µg/plate	14.7
	185.1852 µg/plate	20.7
	555.5556 µg/plate	18.0
	1666.6667 µg/plate	15.7
	5000.0000 µg/plate	15.0
	Positive control	1187.7

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	28.3
	61.7284 µg/plate	28.7
	185.1852 µg/plate	24.7
	555.5556 µg/plate	24.7
	1666.6667 µg/plate	27.7
	5000.0000 µg/plate	23.0
	Positive control	1935.0

 

Strain	Treatment	Mean counts
TA 1537	Negative control	10.3
	61.7284 µg/plate	12.0
	185.1852 µg/plate	14.7
	555.5556 µg/plate	10.0
	1666.6667 µg/plate	9.7
	5000.0000 µg/plate	6.7
	Positive control	2315.0

 

Strain	Treatment	Mean counts
TA 102	Negative control	336.7
	61.7284 µg/plate	340.3
	185.1852 µg/plate	391.7
	555.5556 µg/plate	374.3
	1666.6667 µg/plate	419.3
	5000.0000 µg/plate	357.7
	Positive control	1961.0

 

 

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

Confirmatory experiments with metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	184.7
	61.7284 µg/plate	208.3
	185.1852 µg/plate	226.7
	555.5556 µg/plate	219.3
	1666.6667 µg/plate	226.0
	5000.0000 µg/plate	213.7
	Positive control	1881.0

 

Strain	Treatment	Mean counts
TA 1535	Negative control	17.3
	61.7284 µg/plate	24.3
	185.1852 µg/plate	20.7
	555.5556 µg/plate	16.3
	1666.6667 µg/plate	25.3
	5000.0000 µg/plate	19.7
	Positive control	378.7

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	42.7
	61.7284 µg/plate	43.0
	185.1852 µg/plate	49.3
	555.5556 µg/plate	47.0
	1666.6667 µg/plate	33.7
	5000.0000 µg/plate	33.7
	Positive control	2659.0

 

Strain	Treatment	Mean counts
TA 1537	Negative control	14.7
	61.7284 µg/plate	9.3
	185.1852 µg/plate	12.3
	555.5556 µg/plate	15.3
	1666.6667 µg/plate	9.0
	5000.0000 µg/plate	6.7
	Positive control	414.7

 

Strain	Treatment	Mean counts
TA 102	Negative control	331.7
	61.7284 µg/plate	369.3
	185.1852 µg/plate	379.7
	555.5556 µg/plate	427.3
	1666.6667 µg/plate	496.3
	5000.0000 µg/plate	458.7
	Positive control	1723.3

 

Applicant's summary and conclusion

Conclusions:

FAT 21036/C was not mutagenic in this bacterial reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of FAT 21036/C to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. This test was conducted in accordance with OECD test guideline 471. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

Range in the cytotoxicity test; 20.6, 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate

The concentration range of FAT 21036/C to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the test material was tested for mutagenic effects without and with metabolic activation at five concentrations in the range 61.7, 185.1, 555.5, 1666.6, 5000 µg/plate.

 

Mutagenicity test, original experiment

In the original experiment carried out without metabolic activation, treatment of strain TA 102 with Polar blau RLS roh trocken (FAT 2103 6/C) led to a marginal increase in the number of backmutants at the concentrations of 1666.7 and 5000 µg/plate. No effects were observed with the other strains. In the experiment with activation performed on strain TA 102, a marginal increase in the number of revertants occurred at the concentration of 555.6 µg/plate only. No effects were observed with the other strains.

 

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without microsomal activation, after treatment with Polar blau RLS roh trocken (FAT 21036/C), no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. The effect observed in the original experiment on strain TA 102 could not be reproduced. In the experiment with activation performed on strain TA 102, a marginal increase in the number of back-mutants occurred at the concentration of 1666.7 µg/plate only. Again, no effects were observed with the other strains. In the experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed. A marginal increase in backmutants was reported with strain TA 102 with metabolic activation at 555.6 µg/plate only in the original experiment and at 1666.7 µg/plate only in the confirmatory experiment. This marginal effect seen was regarded as not significant as the increase was seen at two different concentrations during original and confirmatory experiments, without being seen at higher concentrations in these experiments. Thus, the effect seen can not be considered as dose-dependent or concentration driven. Hence, based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 21036/C was not mutagenic in this bacterial reverse mutation assay.