Reaction mass ofDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN1}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]chromate(2-) andDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN2}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]chromate(2-)

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:

Study initiation date: 08 March, 1993; Experiment start date - 19 March, 1993; Experiment end date - 04 May, 1993; Study completion date: 28 June, 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:

yes

Remarks:

a statistical analysis was not performed as no appropriate statistical method is available.

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:

Disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

EC Number:

274-487-5

EC Name:

Disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Cas Number:

70236-55-4

Molecular formula:

C36H18CrN7O13S.2Na

IUPAC Name:

disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification of the test material as used in the study report: Irgalan Schwarz BGL Roh Trocken (FAT 20037/C)
- Source and batch No.of test material: 9300001
- Expiration date of the lot/batch: February 1998

OTHER SPECIFICS:
- Purity: about 80 %

Method

Target gene:

Histidine requiring genes of Salmonella strains

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 obtained from Prof. B. Ames, Berkeley, CA., U.S.A.

Metabolic activation:

with and without

Metabolic activation system:

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 29.4 and 30.6 mg/ml.

Test concentrations with justification for top dose:

Experiment without metabolic activation: 61.7, 185.2, 555.6, 1666.7 & 5000 µg/plate;
Experiment with metabolic activation: 61.7, 185.2, 555.6, 1666.7 & 5000 µg/plate;

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (Suspension)

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:

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:

Without microsomal activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene

Remarks:

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:

Without metabolic 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:

With metabolic activation

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic 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
- Method: Background growth inhibition.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : increase of the mean number of revertants per plate above that of the negative control.

Evaluation criteria:

Assay acceptance criteria:
A test was considered acceptable if the mean colony counts of the control values of all strains were within the acceptable ranges and if the results of the positive controls met the criteria for a positive response. In either case the final decision was based on the scientific judgement of the Study Director.

Criteria for a positive response:
The test substance was 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 as no appropriate statistical method is available.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: Salmonella typhimurium TA 98, TA 100 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the mutagenicity tests without and with metabolic activation, due to toxicity of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest 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:

cytotoxicity

Remarks:

In the mutagenicity tests without and with metabolic activation, due to toxicity of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the mutagenicity tests without and with metabolic activation, due to toxicity of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Six concentrations of FAT 20037/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 were markedly increased at the upper concentrations. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

Any other information on results incl. tables

Mutagenicity test, original experiment

In the original experiment performed without metabolic activation, treatment of strain TA 100 with FAT 20037/C led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. With strain TA 1535 a very weak increase in the number of back-mutants was observed at the concentrations of 1666.7 and 5000 µg/plate. With strain TA 98 a strong, concentration dependent increase in the number of backmutants was observed at all concentrations. With strain TA 1537 a moderate increase in the number of revertants was registered at the concentrations of 61.7 to 555.6 µg/plate. No effect was seen with strain TA 102.

In the original experiment performed with activation, treatment of strain TA 100 with FAT 20037/C led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. With strain TA 1535 a very weak increase in the number of back-mutants was observed at the concentration of 555.6 µg/plate only. With strain TA 98 a strong, concentration dependent increase in the number of back-mutants was observed at the concentrations of 61.7 to 1666.7 µg/plate. With strain TA 1537 a moderate increase in the number of revertants was registered at the concentrations of 61.7 to 1666.7 µg/plate. No effect was seen with strain TA 102. In the experiment with activation performed on strain TA 102 the mean number of colony counts in the negative control was somewhat above the acceptable range given on page 19. This, however, does not affect the test results.

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without metabolic activation, treatment of strain TA 100 with FAT 20037/C again led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. In the confirmatory experiment performed without metabolic activation, treatment of strain TA 100 with FAT 20037/C again led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. The effect observed in the original experiment with strain TA 1535 could not be reproduced. With strain TA 102 a very weak increase in the number of revertants was registerd at the concentration of 555.6 µg/plate. With strain TA 98 a strong, concentration dependent increase in the number of back-mutants was observed at the concentrations of 61.7 to 1666.7 µg/plate . With strain TA 1537 a moderate increase in the number of revertants was registered at the same concentrations.

In the confirmatory experiment performed with activation, treatment of strain TA 100 with FAT 20037/C again led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. With strain TA 1535 a very weak increase in the number of back-mutants was observed at the same concentrations of 185.2 to 5000 µg/plate. With strain TA 98 a strong, concentration dependent increase in the number of back-mutants was observed at the concentrations of 61.7 to 1666.7 µg/plate. With strain TA 1537 a moderate increase in the number of revertants was registered at all concentrations. No effect was seen with strain TA 102. In the mutagenicity tests without and with metabolic activation, due to a growth-inhibiting effect of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Applicant's summary and conclusion

Conclusions:

FAT 20037/C exerted a clear-cut mutagenic action on strains S. typhimurium TA 98, TA 100 and TA 1537.

Executive summary:

The mutagenic potential of the test item was investigated in a bacterial reverse mutation assay according to OECD guideline 471 and EU method B.13/14 in compliance with GLP.

Based on the results of a preliminary toxicity test, FAT 20037/C was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiments was performed with the same concentrations.

In the original experiment performed without and with metabolic activation, after treatment of the bacterial cultures with FAT 20037/C, the number of revertant counts increased strongly with strain TA 98, moderately with strains TA 100 and TA 1537 and slightly with strain TA 1535. In the confimatory experiment performed without activation, after treatment of the bacterial cultures with FAT 20037/C the number of revertant counts again increased strongly with TA 98 and moderately with strains TA 100 and TA 1537. A very weak increase in the number of back-mutants occurred on strain TA 102. The effect observed in the original experiment with strain TA 1535 could not be reproduced. In the confirmatory experiment carried out with activation, a strong increase in the number of back-mutants occurred on strain TA 98, a moderate increase on strains TA 100 and TA 1537 and very weak one on strain TA 1535.

Based on the results of these experiments, it was concluded that FAT 20037/C exerted a clear-cut mutagenic action on strains S. typhimurium TA 98, TA 100 and TA 1537.