Sodium 4-amino-5-hydroxy-3-(4-nitrophenylazo)-6-(phenylazo)naphthalene-2,7-disulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 12th to April 04th, 2000

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:

2000

Materials and methods

Test guideline

Qualifier:

according 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:

Sodium 4-amino-5-hydroxy-3-(4-nitrophenylazo)-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

213-903-1

EC Name:

Sodium 4-amino-5-hydroxy-3-(4-nitrophenylazo)-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

1064-48-8

Molecular formula:

C22H16N6O9S2.2Na

IUPAC Name:

sodium 4-amino-5-hydroxy-3-(4-nitrophenylazo)-6-(phenylazo)naphthalene-2,7-disulphonate

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The bacterial strains TA 1535, TA98 and TA 100 were obtained from Dr. B.N. Ames (University of California, Berkley). The bacterial strains TA 1537 was obtained from BASF. The bacterial starin WP2 uvrA was obtained from Dr. Heinz Trager, Knoll AG.
Regular checking of the properties of the stains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to Ames et al.
The stains cultures were stored as stock cultures in ampoules with nutrient broth + 5% DMSO (MERCK, D-Darmstadt) in liquid nitrogen.
From the thawed ampoules of the strains 0.5 ml bacterial suspension were transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin (25 µg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK)
5 g NaCl (MERCK)
The bacterial culture was incubated in a shaking water bath for 8 hours at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Pre-Experiment and Mutation Experiment 1: 33; 100; 333; 1000; 2500 and 5000 µg/plate
Mutation Experiment 2: 33; 100; 333; 1000; 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle: deionised water.

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene; 4-Nitro-o-phenylene-diamine

Remarks:

Without S9: NaN3 for TA100 and TA 1535, 4-NOPD for TA 1537 and TA 98, MMS for WP2uvrA. With S9: congo red for TA 98, 2-AA for all the other strains.

Details on test system and experimental conditions:

PRE-EXPERIMET
To evaluate the toxicity of the test item a prestudy was performed with strins TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA.Eight concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described for the experiment I.
The per-experiment will be reported as the main experiment I if the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more.
Toxicity of the test item results in a reduction in the number of spontaneous revertants a clearing of the bacterial background lawn.

DOSE SELECTION
In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as part of the experiment I since no relevant toxic effects was observed and 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
Experiment I and II: 33; 100; 333; 1000; 2500; 5000 µg/plate.

EXPERIMENTAL PERFORMANCE
For each strain and dose level, including the controls three plates were used.
The following materials were mixed in a test tube and incubated al 30 °C for 30 min.
100 µl Test solution at each dose level, solvent ( negative control) or reference mutagen solution (positive control)
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl bacterial suspension (cf. test system, pre-culture of the strains)
2000 µl Overlay agar
After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DATA RECORDING
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation BIOSYS GmbH). Due to the intense colour of the test article the colonies were counted manually at higher concentratuons in experiment II.

ACCEPTABILITY OF THE ASSAY
The assay was considered acceptable if it meets the following criteria:
- Regular background growth in the negative and solvent control
- The spontaneous reversion rates in the negative , solvent and positive control are in the range of our historical data
- The positive control substances should produce a significant increase in mutant colony frequencies.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
The S9 liver microsomal fraction was obtained from the liver of 7-8 weeks old male Syrian golden hamsters ( BRL).
After decapitation of the anaesthetized animals the livers of the animals were removed , washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in deionized water and homogenized. The homogenate, diluted 1+3 in sodium phosphate buffer was centrifuged at 9,000 g for 10 minutes at 4 °C.
A stock of supernatants containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules were kept at -20 °C for up to one week before use. The protein content is determined using an analysis kit of Bio-Rad Laboratories . the protein concentration of S9 preparation was 27.4 mg/ml (lot. No. 270899) in all experiments.

HAMSTER S9 MIX
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant is 30% v/v. the concentrated cofactor solution yields the following concentrations in the S9 mix.
8.0 mM MgCl2
33.0 mM KCl
20.0 mM Glucose-6-phosphate
2.8 units/ml Glucose-6-phosphate-dehydrogenase
4.0 mM NADP
2.0 mM NADH
2.0 mM FMN
In 100 ml mM Sodium-Ortho-Phosphate-buffer, pH 7.4
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. and Prival and Mitchell.

Evaluation criteria:

a test item is considered as positive if a biologically relevant and dose related incease in the number of is induced. A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows: A test item is considered as mutagenic if the number of reversion is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and WP2 uvrA, or thrice in TA 1535 and TA 1537.
Also a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose will induce the criteria described above or not.

Statistics:

No statistical evaluation of the data is required

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

True negative controls validity:

not valid

Positive controls validity:

not valid

Additional information on results:

The assay was performed in two independent experiment, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.
No relevant toxic effects, evident as a reduction in the number of revertants, was observed in any of the strains up to the maximal concentration.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S) mix in all strains used.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Acid black 1 at any dose level, neither in the presence nor absence of metabolic activation (S) mix). There was also no dose dependent increase of the mutation rates in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

The substance did not induce gene mutations under test conditions.

Executive summary:

This study was performed to investigate the potential of Acid Black 1 to induce gene mutation according to the pre-incubation test using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and the Escherichia coli WP2 uvrA.

The assay was performed in two independent experiment, 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:

33; 100; 333; 1000; 2500 and 5000 µg/plate.

 

No relevant toxic effects, evident as a reduction in the number of revertants, was observed in any of the strains up to the maximal concentration.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S) mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Acid black 1 at any dose level, neither in the presence nor absence of metabolic activation (S) mix). There was also no dose dependent increase of the mutation rates in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

 

CONCLUSION

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by frameshifts or base exchanges in the genome of any of the strains used.