2,4-bis(((2-(dimethylammonio)ethyloxy)carbonyl)phen-2-ylazo)benzene-1,3-diolbis(methanesulfonate)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): Basazol Gelb 8511
- Physical state: powder
- Lot/batch No.: ZD 00852/046/02
- Storage condition of test material: room temperature

Method

Target gene:

Salmonella typhimurium: (his-)
E. coli: (Trp-)

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S-9 mix

Test concentrations with justification for top dose:

4µg - 5000 µg/plate (SPT; Salmonella strains); 20 µg - 5000 µg/plate(SPT; E. coli)
4µg - 1000 µg/plate (PIT; TA 1535, TA 98); 4µg - 1500 µg/plate (PIT; TA 100, TA 1537)
4µg - 2500 µg/plate (PIT; E. coli)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was selected as the vehicle.

Controls

Controls 1

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537;without S9-mix

Controls 2

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenenylenediamine

Remarks:

TA98; without S9-mix

Controls 3

Positive controls:

yes

Positive control substance:

other: N-methyl-N´-nitro-N-nitrosoguanidine

Remarks:

TA100 and TA1535; without S9-mix

Controls 4

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

E.coli WP2 uvrA; without S9-mix

Controls 5

Untreated negative controls:

yes

Remarks:

Carried out in order to check for possible contaminants (sterility control).Additional plates are treated with soft agar S-9 mix, buffer, vehicle or the test substance but without the addition of tester strains

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

same concentration and volume for all tester strain in order to determine the spontaneous mutation rate

Details on test system and experimental conditions:

Standart plate test
Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6%; agar + 0.6%; NaCl) and 10 ml amino acid solution (minimal
amino acid solution for the determination of mutants: 0.5 mM histidine+ 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining
components are added in the following order:
0.1 ml test solution
0.1 ml fresh bacterial culture
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolicactivation)

After mixing, the samples are poured onto the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of about 20 minutes using a
shaker.S ubsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.

Both tests
In each experiment 3 test plates per dose or per control used; after incubation at 37°C for 48 hours in the dark, the bacterial colonies (his* revertants) are counted.
After incubation at 37°C for 48 - 72 hours in thedark, the bacterial colonies (his+ revertants), trp+ revertants) arecounted.

The titer is generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest
doses in all experiments.

Evaluation criteria:

The test chemical is considered positive in this assayi f the following criteria are met:
A dose-related and reproducible increase in the number 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 adding 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 experimentscarried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium strains TA 100 and TA 1537, E. coli WP2 uvrA in PIT

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: all strains tested in SPT and PIT

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Standard Plate Test (SPT):

Tests without S-9 mix: TA1535, TA 100, TA1537, TA98, E. coli WP2 uvrA: No increase in the number of his+ or trp+ revertants.

Tests with S-9 mix: TA1535, TA 100, TA1537, TA98, E. coli WP2 uvrA: No increase in the number of his+ or trp+ revertants.

 

Preincubation Test (PIT):

Tests without S-9 mix: TA1535, TA 100, TA1537, TA98, E. coli WP2 uvrA: No increase in the number of his+ or trp+ revertants.

Test with S-9 mix: TA 1535, TA98: No increase in the number of his+ revertants; TA 100: Slight increase in the number of his+ revertants over a dose range of about 500 µg - 1500 µg/plate (factor 1.3 - 2.3); TA 1537: increase in the number of his+ revertants from about 500 µg- 750 µg/plate (factor 2.4 - 3.0 onward with a maxium at 1000 µg- 1250 µg/plate (factor 3.6 -4.4); E.coli WP2 uvrA: Weakly positive reaction of about 500µg/plate (factor 2.0).

 

Cytotoxicity:

A bacteriotoxic effect (reduced his+ or trp+ background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions at doses from about 500 µg - 2500 µg/plate onward (SPT).

In the preincubation assay bacteriotoxicity was found without S-9 mix from about 100µg - 500µg/plate onward (all tester strains) or with S-9 mix depending on the strain at doses >= 1,000µg/plate (TA 1535, TA1537, TA 98, E. coli WP2 uvrA).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Thus, under the experimental conditions chosen here, it is concluded that Basazol Gelb 8511 is a mutagenic agent in a bacterial reverse mutation test in vitro.