4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

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:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Ames, B. N.; McCann, J.; Yamasaki, E.: Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mut. Res. 31 (1975) 347-364.

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

EC Number:

235-436-2

EC Name:

4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

Cas Number:

12227-50-8

Molecular formula:

C54H32N4O6

IUPAC Name:

4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

Test material form:

solid: particulate/powder

Details on test material:

Vat Yellow 33

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Test Concentrations for preliminary toxicity test & mutation study:
0.8, 4, 20, 100, 500 μg/well
The test product was found to have a maximum solubility of approximately 5,000 μg/ml but at levels of 500 μg/well and below was found to be non-toxic to the tester strains.
Accordingly, dose levels ranging from 0.8 μg/plate to 500 μg/plate were selected for the mutation study.

Vehicle / solvent:

For the purpose of this study the test material was dissolved/suspended in Dimethyl sulphoxide.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: M-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG); 4 Nitro-O-phenyldiamine (4NOPD); 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

Bacteria
The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening.

TA 1535 - sensitive to agents inducing base-pair substitution
TA 100 - sensitive to agents inducing base-pair substitution
TA 1537 - sensitive to agents inducing frame-shift mutations
TA 98 - sensitive to agents inducing frame-shift mutations

The bacteria were obtained from Professor B Ames (Department of Biochemistry, University of California, Berkeley, California, U.S.A.). Overnight cultures of these specimens were used to generate Master slopes which were maintained at 4°C for use as routine sources of inoculum for cultures to be used in this assay.
Prior to being used in this test characterisation checks were carried out on each strain to determine (i) histidine requirement (ii) crystal violet sensitivity (iii) presence of R factor, and (iv) spontaneous reversion rate.
In this assay overnight sub-cultures of the Master slopes were prepared in nutrient broth (Oxoid Ltd) incubated at 37 °C for approximately 18 hours.
These overnight cultures yielded approximately 10E+8 – 10E+9 bacteria per ml and were used as the standard bacterial suspension.

Microsomal Enzyme Fraction
A commercial preparation of this enzyme fraction was obtained from Uniscience Limited, 8 Jesus Lane, Cambridge, CB5 8BA. This fraction is obtained from animals pre-treated with Arochlor 1254 (a mixture of polychlorinated biphenyls) and was stated to be from batch no. 03121 protein level 25 mg/ml.

Test Procedure
a) Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test compound to the tester organisms. 0.1 ml of bacterial suspension was added to 4 ml of histidine deficient media (histidine & top agar) and overlayed onto sterile plates of Vogel-Bonner agar (minimal agar - 15 ml/plate). When the agar had set, wells were made on each plate and 0.1 ml aliquots of concentrations ranging from 8 μg/ml to 5,000 μg/ml of test material were added to each well. These plates were incubated at 37 °C for 24 hours after which time the toxicity of the test material was assessed by measuring the zones of inhibition around each well.
Dimethyl Sulphoxide which was used as a solvent diluent for the test material in this assay was also added to each plate as a control.

Mutation Study
Four concentrations of the test material were assayed in triplicate against each tester strain, with and without metabolic activation.
0.1 ml of the appropriately diluted test material, negative or positive control solution were placed in sets of sterile universal bottles containing 9 ml of molten histidine deficient top agar at 45 °C, these sets comprising two bottles for each tester strain/test material or control solution. A 0. 1 ml aliquot of one of the bacterial suspensions was added to each of the bottles. Into one of the duplicate sets of bottles was placed 0 .5 ml of the S-9 liver microsone mix; in the other set the S-9 mix was omitted. The contents of the bottle were then mixed and a 3 ml aliquot of each poured on top of Vogel-Bonner agar plates.
These plates were then incubated at 37 °C for 48 hours and the number of revertant colonies counted.

Rationale for test conditions:

In accordance with the test procedures.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than two fold at all dose levels employed, the intervals of which should be between 3 and 5 fold and extend to the limits imposed by toxicity or solubility.

Results and discussion

Test results

Test results 1

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

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Test results 3

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

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity study
The test product was found to have a maximum solubility of approximately 5,000 μg/ml but at levels of 500 μg/well and below was found to be non-toxic to the tester strains.
Accordingly, dose levels ranging from 0.8 μg/plate to 500 μg/plate were selected for the mutation study.

Mutation study
The overnight culture of each strain was found to be in the required range of 10E+8 to 10E+9 bacteria/ml and the spontaneous reversion rate for each was found to be within the expected range.
No significant increase in the number.of revertant colonies was recorded for any of the strains at any of the dose levels employed in this study, either with or without metabolic activation. All counts of revertant colonies were similar to those recorded for the negative control plates and were within the range expected for spontaneous reversion for each strain.

The positive control substances all produced significant increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Applicant's summary and conclusion

Conclusions:

Vat Yellow 33 was found to exhibit no evidence of mutagenic activity under the conditions of this experiment

Executive summary:

This study was conducted according to Safepharm Standard Test Method M26 and was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers and Garner et al in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test compound in the presence and absence of S9-mix from induced rat livers.. The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening: TA 1535, TA 100, TA 1537, TA 98.

In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test compound to the tester organisms. In the main study, four concentrations of the test material were assayed in triplicate against each tester strain, with and without metabolic activation.

In the preliminary toxicity study, the test substance was found to have a maximum solubility/suspendability of approximately 5,000 μg/ml. At levels of 500 μg/well and below, the test substance was found to be non-toxic to the tester strains. Accordingly, dose levels ranging from 0.8 μg/plate to 500 μg/plate were selected for the mutation study.

I the main study, the overnight culture of each strain was found to be in the required range of 10E+8 to 10E+9 bacteria/ml and the spontaneous reversion rate for each was found to be within the expected range. No significant increase in the number of revertant colonies was recorded for any of the strains at any of the dose levels employed in this study, either with or without metabolic activation. All counts of revertant colonies were similar to those recorded for the negative control plates and were within the range expected for spontaneous reversion for each strain. The positive control substances all produced significant increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Consequently, the test substance was found to exhibit no evidence of mutagenic activity under the conditions of this experiment.